Polycistronic expression system for bacteria

ABSTRACT

The invention relates to polycistronic expression in gram-positive bacterium and in particular concerns polycistronic expression units comprising one or more gene endogenous to the gram-positive bacterium transcriptionally coupled to one or more genes exogenous to the bacterium.

RELATED APPLICATIONS

This is a 371 of PCT application No. PCT/EP2012/060431, filed Jun. 1,2012, which claims priority from EP 11168495.7 filed Jun. 1, 2011 and EP11173588.2 filed Jul. 12, 2011, both of which are incorporated herein byreference in their entireties.

The material in the ASCII text file, named DCIP-51911-Seq-Listing.txt,created Nov. 18, 2013, file size 4096 bytes, is hereby incorporated byreference.

FIELD OF THE INVENTION

The invention belongs to the fields of biology and medicine, moreparticular molecular and cellular biology, and relates to recombinantengineering and expression of products such as peptides, polypeptides orproteins by microorganisms. More specifically, the invention relates topolycistronic expression constructs or cassettes for expression of suchproducts by microorganisms, and further to related vectors, transformedhosts, uses and applications, such as delivery, especially therapeuticdelivery, of so-expressed products to subjects.

BACKGROUND OF THE INVENTION

To date, many expression systems for recombinant proteins have beendeveloped, for various biotechnological applications. Systems forheterologous or homologous gene expression have been established inprokaryotes, yeasts and fungi and in mammalian cells.

Most recombinant proteins produced in yeasts have been expressed usingSaccharomyces cerevisiae as the host system. Despite this, severallimitations have been detected in the S. cerevisiae system. Examples areproduct yield, which is usually low, and inefficient secretion (many S.cerevisiae proteins are not found free in the culture medium but ratherare retained in the periplasmic space or associated with the cell wall)(Dominguez et al. Int. Microbiol., 1998, vol. 1(2), 131-142). Because oflimitations of production in yeast, a lot of interest arose forexpression of proteins in bacteria, which are easy to grow in aninexpensive broth and are frequently used to produce recombinantproteins. Among prokaryotic systems, the highest protein levels areusually obtained using recombinant expression in Escherichia coli (E.coli) (Jana & Deb. Appl. Microbiol. Biotechnol., 2005, vol. 67(3),289-298). However, in E. coli, the most commonly used productionstrategies are intracellular (in the periplasm or cytoplasm), andtherefore involve expensive and often problematic downstreampurification processes.

Lactic Acid Bacteria (LAB) are becoming increasingly important as hostsfor recombinant expression of heterologous polypeptides in vitro (e.g.,U.S. Pat. No. 5,559,007), as well as for in vivo or in situ expressionand delivery of antigens and/or therapeutically relevant polypeptides(e.g., WO 97/14806). Heterologous proteins produced in theseGram-positive bacterial hosts can easily be secreted into the medium,thus facilitating their purification as well as their direct delivery tosubjects.

Most expression systems can handle very well the expression of onesingle protein (as a result of one single gene sequence). However, insome cases it is desirable to have an expression system that is capableof expressing multiple proteins or multigenic protein complexes, forexample, the in vitro expression of antibodies or protein complexes, butalso in vivo or in situ expression and delivery of two or more proteinsthat have a synergistic effect in a particular disease or the in vivo orin situ expression and delivery of antibodies or functional (multigenic)fragments thereof. In these cases, it is desirable to have the multiplegenes that are encoding the desired proteins or antibodies under thecontrol of one promoter, because of the necessity of tight co-regulationof the multiple genes.

The two most common approaches to produce recombinant protein complexesare to perform in vitro reconstitution of individually expressed andpurified subunits, or to implement in vivo reconstitution byco-expressing the subunits in an appropriate host (Selleck & Tan,“Recombinant protein complex expression in E. coli”, Curr. Protoc.Protein Sci., 2008, chapter 5:unit 5, 21). Although in vitroreconstitution has been successfully used, the process is tedious (eachsubunit has to be expressed and purified, and the complex has to befurther purified after reconstitution) and reconstitution yields areoften low. In contrast, in vivo reconstitution by co-expression offersthe benefits of efficiency (only one round of expression andpurification) and potentially higher yields and quality of the desiredcomplex (refolding and assembly of the complex take place in thepresence of protein folding enzymes in a cellular environment) (Selleck& Tan 2008, supra). In vivo reconstitution has been successfullyperformed by co-infecting insect cells with baculoviruses expressingindividual protein subunits (Tirode et al. Mol. Cell, 1999, vol. 3(1),87-95), and in bacteria from multiple plasmids (Johnston et al. ProteinExpr. Purif., 2000, vol. 20(3), 435-443; McNally et al. Proc. Natl.Acad. Sci. USA, 1988, vol. 85(19), 7270-7273) or from specializedpolycistronic plasmids (Henricksen et al. J. Biol. Chem., 1994, vol.269(15), 11121-11132; Ishiai et al. J. Biol. Chem. 1996, vol. 271(34),20868-20878; Li et al. Proc. Natl. Acad. Sci. USA, 1997, vol. 94(6),2278-2283).

General polycistronic expression systems for producing protein complexesin E. coli have been described (Selleck & Tan 2008, supra; Tan. Protein.Expr. Purif., 2001, vol. 21(1), 224-234; Tan et al. Protein Expr.Purif., 2005, vol. 40(2), 385-395). These systems utilize the concept ofa translation cassette, comprised of the coding region with requisiteSTART and STOP codons and preceded by translational initiation signalssuch as the Shine-Dalgarno (SD) sequence and translational enhancers(Tan 2001, Tan et al. 2005, supra). When transcribed into mRNA, thetranslation cassette contains the necessary and sufficient informationfor the E. coli translational machinery to initiate and sustaintranslation of the mRNA into the desired polypeptide (Selleck & Tan2008, supra).

A bi-cistronic expression vector for interleukin-18 has been describedin E. coli, however, the intergenic region between the two genesconsisted of a synthetic linker, and is clearly gene-specific as theexpression of the caspase-4 was much higher than the expression of ICE.Smolke et al. previously demonstrated that it is possible todifferentially control the protein levels encoded by two or more genesin an operon using synthetic intergenic region sequences (Smolke et al.Appl. Environ. Microbiol., 2000, vol. 66(12), 5399-5405; Smolke &Keasling. Biotechnol. Bioeng., 2002, vol. 80(7), 762-776). However, thisapproach relies on random combinations, and requires the introduction ofsynthetic sequences into the expression host.

The demand for new and improved antibody production systems has arisenin recent years. Systems for antibody expression have been establishedin prokaryotes, yeasts and fungi and in mammalian cells. Although singlechain and single domain antibodies are easier to produce from bacteria,full-size antibodies generally have higher binding affinities and lessrisk for formation of neutralizing antibody when injected.

Full-size antibodies can be produced from bacteria (Mazor et al. Nat.Biotechnol., 2007, vol. 25(5), 563-565; Simmons et al. J. Immunol.Methods, 2002, vol. 263(1-2), 133-147). Most reports on recombinantprokaryotic expression describe production of antibody fragments, albeitalmost exclusively from E. coli. Although many engineered LAB arecapable of correct disulphide bonding, the literature contains only alimited number of examples of antibody-like molecules produced from LAB(Kruger et al. Nature Biotechnology, 2002, vol. 20(7), 702-706; Beninatiet al. Nature Biotechnology, 2000, vol. 18(10), 1060-1064; Chancey etal. J. Immunol., 2006, vol. 176(9), 5627-5636; Hultberg et al. BMCBiotechnol., 2007, vol. 7, 58; Yuvaraj et al. Mol. Nutr. Food. Res.,2008, vol. 52(8), 913-920). These reports only describe single chainantibody fragments expressed in Lactobacillus species, Lactococcuslactis and Streptococcus gordonii, and not multigenic, double chainantibody fragments or full-sized antibodies.

Polycistronic expression systems could be crucial in obtaining efficientprokaryotic synthesis and expression of complex proteins such asantibodies. Since the FDA approval in 1986 of Muromonab-CD3, still oneof the most potent immunosuppressive drugs available for the managementof transplant rejection (Hooks et al. Pharmacotherapy, 1991, vol. 11(1),26-37), full-size antibodies and antibody fragments have becomeincreasingly important and versatile tools in medicine.

While the current state of the art reveals several examples ofpolycistronic expression systems in bacterial cells, these are quitelimited, highlighting the need for a more efficient system forintroducing and expressing multiple genes. Accordingly, there exists aneed to provide further sequences which can be favourably used forexpression of proteins, preferably heterologous protein expression andeven more preferably multiple heterologous protein expression.

In addition to the above, the endeavour to produce higher amounts ofrecombinant protein, both for direct protein delivery by recombinantmicroorganisms as well as for bulk protein production and down streampurification, represents a great technological strive. An existingapproach to increase production of heterologous proteins is the use ofselected strong promoters (see for instance WO 2008/084115). In thisapproach, proteomic analysis is performed to identify the most abundantendogenous proteins expressed by a microorganism. By use of the genomesequence, the respective genes and promoters can be identified andisolated. These strong promoters (e.g. the Lactococcus lactis hIIA genepromoter, PhIIA) can be positioned in front of a heterologous gene andin this way, high expression can be achieved. However, a level ofexpression which impairs host physiology may impose a growth burden onthe host and results in counter-selection. This intrinsically limits thehighest possible expression of any given heterologous protein in anexpression host to a certain specific level. This is an especiallycumbersome obstacle in the development of chromosomally locatedexpression units.

The issue of counter-selection is traditionally addressed by theprovision of selection markers. Indeed, positive or negative selection,e.g. by providing antibiotic resistance genes, can prevent loss of theintroduced heterologous gene. Alternatively, or in addition to the useof selection markers, inducible gene expression systems may be employed,which allow for uncoupling propagation of the host and expression of theheterologous protein, thereby preventing possible counter-selectionduring the propagation phase when the heterologous gene is notexpressed. In this context, EP0569604 describes an inducible expressionsystem in Streptococcus thermophilus in which a heterologous gene isobligatory positioned 5′ to the LacZ gene. In this way, expression ofthe heterologous gene is not only inducible but in addition maintenanceof the heterologous gene is also selected for by growing the bacteria intheir natural habitat, milk, with lactose as carbon source, whichrequires the expression of the LacZ gene.

It is clear that the systems for heterologous gene expression describedabove are limited in application. For instance, the use of selectionmarkers, such as antibiotic resistance genes, is not readily toleratedfor applications in food production or in pharmaceutical applications.Further, limitation to growing in the natural habitat or to using thecarbon source from the natural habitat for growth significantly reducesthe versatility of any system for heterologous gene expression. Also,the use of inducible systems is inherently dependent on the growthconditions of the host, such that defined culture media, to which aninducer is to be added, are needed to ensure expression of theheterologous protein.

Accordingly, there also exists a need in the art to increaseheterologous protein expression; and sequences, cloning systems andstrategies are needed which can achieve high expression levels in orderto obtain sufficient amounts of expressed heterologous proteins inindustrial and/or therapeutic settings, while at the same time beingversatile and widely applicable under a variety of different conditions.In these settings it would also be particularly useful to obtainexpression of multiple proteins, each having its own biological activityand therapeutic effect.

SUMMARY OF THE INVENTION

The aspects and embodiments of the present invention address at leastsome, e.g., one or more, of the above discussed needs of the art.

The inventors have surprisingly found that gram-positive bacteria canefficiently express exogenous or heterologous genes from polycistronicexpression units also comprising endogenous gene(s) of these bacteria.Thus, gram-positive bacteria can efficiently express exogenous orheterologous genes from polycistronic expression units when such genesare transcriptionally or translationally coupled to endogenous gene(s)of these bacteria. Unexpectedly, the inventors have found thattranscriptional and/or translational coupling of endogenous genes andexogenous genes in polycistronic expression units results in highexpression levels of the exogenous genes in gram-positive bacteria. Inparticular, expression levels of exogenous genes transcriptionallyand/or translationally coupled to gram-positive bacterial endogenousgenes were found to be at least comparable to and advantageously higherthan expression levels of exogenous genes which are nottranscriptionally or translationally coupled to gram-positive bacterialendogenous genes.

Accordingly, in an aspect the invention relates to a gram-positivebacterium comprising a polycistronic expression unit, said polycistronicexpression unit comprising one or more endogenous genes and one or moreexogenous genes. The polycistronic expression unit can thus also bedenoted as comprising an endogenous gene (for example but withoutlimitation one endogenous gene) and one or more exogenous genes.Preferably, the polycistronic expression unit consecutively comprisesone or more endogenous genes and one or more exogenous genes. Suchpolycistronic expression unit can thus also be denoted as consecutivelycomprising an endogenous gene (for example but without limitation oneendogenous gene) and one or more exogenous genes. The polycistronicexpression unit is configured to effect transcription of the one or moreendogenous genes and the one or more exogenous genes in a polycistronicmRNA. Hence, the present gram-positive bacterium may otherwise bedenoted as comprising one or more endogenous genes to which one or moreexogenous genes are transcriptionally or translationally coupled. Alsoprovided is thus a gram-positive bacterium comprising one or moreendogenous genes to which one or more exogenous genes aretranscriptionally and/or translationally coupled.

Another aspect provides a recombinant nucleic acid comprising apolycistronic expression unit, said polycistronic expression unitcomprising a gene endogenous to a gram-positive bacterium and one ormore genes exogenous to the gram-positive bacterium. Preferably, thepolycistronic expression unit consecutively comprises one or moreendogenous genes and one or more exogenous genes. Hence, also providedis a recombinant nucleic acid comprising a polycistronic expression unitcomprising one or more gene endogenous to a gram-positive bacterium towhich one or more genes exogenous to the gram-positive bacterium aretranscriptionally and/or translationally coupled.

Preferably, as intended throughout this specification said one or moreexogenous genes may be transcriptionally or translationally coupled tothe 3′ end of said one or more endogenous genes. The inventors havesurprisingly found that such configuration is beneficial in respect ofheterologous protein expression levels, maintenance and/or genomicstability of the polycistronic expression unit. It has moreover beenfound that further downstream genomic arrangement is of lesser or noimportance.

The transcription of the transcriptionally or translationally coupledone or more endogenous genes and one or more exogenous genes may besuitably regulated or controlled by a promoter capable of achievingtranscription in the gram-positive bacterium, and preferably may beregulated or controlled by an endogenous promoter of said gram-positivebacterium. Hence, also provided is a gram-positive bacterium comprisingone or more endogenous genes located in its native chromosomal locus, towhich one or more exogenous genes are transcriptionally ortranslationally coupled. Preferably, transcription of thesetranscriptionally or translationally coupled one or more endogenousgenes and one or more exogenous genes are thus controlled or regulatedby the native promoter of said one or more endogenous genes. Suitably,the transcriptional or translational coupling may be achieved bychromosomally integrating the one or more exogenous genes to said locus,such as for example by chromosomally integrating the one or moreexogenous genes 3′ of said one or more endogenous genes in said locus.

Accordingly, in an aspect, the invention relates to a recombinantnucleic acid or a gram-positive bacterium comprising a polycistronicexpression unit, said polycistronic expression unit consecutivelycomprising an endogenous gene and one or more exogenous genestranscriptionally coupled to the 3′ end of said one or more endogenousgene, preferably wherein said one or more exogenous gene(s) is (are) themost 3′ gene(s) of the polycistronic expression unit.

The inventors have surprisingly found that chromosomal integration of anexogenous or heterologous gene (or multiple heterologous genes)transcriptionally coupled 3′ to a native gene, which in itself may be apolycistronic gene, such as for instance an operon, yields a stableexpression unit, in which counter selection against the (one or more)exogenous gene is absent or minimal, contrary to expectations.

The inventors have unexpectedly found that the herein describedadvantages are increasingly manifested when the expression of thepolycistronic expression unit is effected under certain conditions, inparticular by certain types of promoters. The inventors havesurprisingly found that polycistronic expression systems as describedherein, in which counter-selection against the heterologous protein(s)is not addressed by conventional measures, such as the use of selectionmarkers, or by the use of inducible systems, may nevertheless be stablymaintained and expressed at high levels, thereby being broadlyapplicable under a variety of different conditions, absent the need ofselection agents or inducers. The polycistronic expression modules asdescribed herein thus allow the use of non-selectable endogenous and/orexogenous genes.

In an aspect, the invention relates to a recombinant nucleic acid or agram-positive bacterium comprising a polycistronic expression unit, saidpolycistronic expression unit consecutively comprising an endogenousgene and one or more exogenous genes transcriptionally coupled to the 3′end of said endogenous gene, wherein expression of said polycistronicexpression unit is effected by a constitutive promoter.

In another aspect, the invention relates to a recombinant nucleic acidor a gram-positive bacterium comprising a polycistronic expression unit,said polycistronic expression unit consecutively comprising anendogenous gene and one or more exogenous genes transcriptionallycoupled to the 3′ end of said endogenous gene, wherein expression ofsaid polycistronic expression unit is effected by a central metabolismgene promoter.

In another aspect, the invention relates to a recombinant nucleic acidor a gram-positive bacterium comprising a polycistronic expression unit,said polycistronic expression unit consecutively comprising anendogenous gene and one or more exogenous genes transcriptionallycoupled to the 3′ end of said endogenous gene, wherein expression ofsaid polycistronic expression unit is effected by a housekeeping genepromoter.

In another aspect, the invention relates to a recombinant nucleic acidor a gram-positive bacterium comprising a polycistronic expression unit,said polycistronic expression unit consecutively comprising anendogenous gene and one or more exogenous genes transcriptionallycoupled to the 3′ end of said endogenous gene, wherein expression ofsaid polycistronic expression unit is effected by an essential genepromoter.

In another aspect, the invention relates to a recombinant nucleic acidor a gram-positive bacterium comprising a polycistronic expression unit,said polycistronic expression unit consecutively comprising anendogenous gene and one or more exogenous genes transcriptionallycoupled to the 3′ end of said endogenous gene, wherein expression ofsaid polycistronic expression unit is not effected by an inducible genepromoter.

In another aspect, the invention relates to a recombinant nucleic acidor a gram-positive bacterium comprising a polycistronic expression unit,said polycistronic expression unit consecutively comprising anendogenous gene and one or more exogenous genes transcriptionallycoupled to the 3′ end of said endogenous gene, wherein expression ofsaid polycistronic expression unit is effected by a ribosomal genepromoter.

In another aspect, the invention relates to a recombinant nucleic acidor a gram-positive bacterium comprising a polycistronic expression unit,said polycistronic expression unit consecutively comprising anendogenous gene and one or more exogenous genes transcriptionallycoupled to the 3′ end of said endogenous gene, wherein expression ofsaid polycistronic expression unit is effected by a glycolysis genepromoter.

As also indicated above, in a preferred embodiment, the promoters asdescribed above are endogenous gene promoters. As is also detailedfurther below, preferably, the promoters as used herein are strongpromoters. Preferably but without limitation, said endogenous promotermay be selected from the group consisting of the promoters of eno,usp45, gapB, pyk, rpmB, and rplS. Very preferably, the transcription ofthe translationally coupled endogenous gene and one or more exogenousgene may be regulated or controlled by the native promoter of (one of)said endogenous gene.

It is to be understood that the characteristics of the promoters asdescribed herein may be combined according to the invention.Accordingly, in embodiments, the invention relates to a recombinantnucleic acid or a gram-positive bacterium comprising a polycistronicexpression unit, said polycistronic expression unit consecutivelycomprising an endogenous gene and one or more exogenous genestranscriptionally coupled to the 3′ end of said endogenous gene, whereinexpression of said polycistronic expression unit is effected by forinstance a (endogenous) constitutive housekeeping gene promoter, a(endogenous) constitutive central metabolism gene promoter, a(endogenous) constitutive essential gene promoter, a (endogenous)constitutive ribosomal gene promoter, a (endogenous) constitutiveglycolysis gene promoter, a (endogenous) central metabolism housekeepinggene promoter, a (endogenous) essential central metabolism genepromoter, a (endogenous) essential central metabolism housekeeping genepromoter, a (endogenous) essential housekeeping gene promoter, a(endogenous) constitutive central metabolism housekeeping gene promoter,a (endogenous) constitutive essential central metabolism housekeepinggene promoter, a (endogenous) essential ribosomal gene promoter, a(endogenous) essential glycolysis gene promoter, a (endogenous)constitutive essential ribosomal gene promoter, a (endogenous)constitutive essential glycolysis gene promoter.

Preferably as intended throughout this specification said one or moreexogenous genes may be transcriptionally or translationally coupled tothe 3′ end of said one or more endogenous genes whereby the one or moreendogenous genes are present at their native position on the bacterialchromosome. In this configuration, the sequence at the 5′ end of the oneor more endogenous genes (minimally including the endogenous genespromoter) is identical to that of the wild type strain and the regionsubsequent to the 3′ end of the one or more exogenous genes areidentical to the sequence of the region 3′ of the one or more endogenousgenes as in the wild type strain.

Many applications of exogenous protein expression such as for instancefor therapeutic protein delivery by recombinant microorganisms canbenefit from the expression of said therapeutic protein in specificselected host microorganisms. These microorganisms could be selectedbased on their colonizing capacity, as e.g. selected strains originatingfrom the human or animal microbiota. Microorganisms could also beselected on their capacity to potentiate the activity of any specificdelivered therapeutic protein e.g. as a consequence of the interactionof their cell wall, cell surface or intracellular content with the hostimmune system e.g. through interaction with toll like receptors, Igfamily members, complement, cytokines and other. Specific microorganismscould be selected for their robustness to persist in or on specificharsh delivery sites, such as intratumoural, skin, sites with high bilecontent, sites with low pH and other. The gram-positive bacterium asrecited throughout this specification may be preferably a lactic acidbacterium (LAB), more preferably a Lactococcus sp., even more preferablyLactococcus lactis or a subspecies or strain thereof. Alternatively,said LAB may be preferably an Enterococcus sp., more preferablyEnterococcus fecium or Enterococcus faecalis or a subspecies or strainthereof.

To avoid lateral gene transfer to endogenous microflora, expression froma chromosomally embedded expression unit is highly favourable for use ofrecombinant microflora as delivery tools for therapeutic proteins inmedicine. Also, chromosomally located expression units may prove to bemuch more stably inherited over generations, so that chromosomallylocated expression units may be the desirable structure for productionstrains used in bulk protein production. In the current state of theart, chromosomal insertion is performed by use of knock-in (KI) typevectors which are conditionally non-replicative and which contain theheterologous gene in-between flanking regions that allow homologousrecombination. In a conventional approach (see for instance WO2008/084115), the KI plasmid is constructed in the homologous host (KIplasmid for L. lactis is built in L. lactis). This is especially thecase for heterologous expression that requires protein secretion, asmany secretion signals are not compatible for use in other hosts. Theuse of strong promoters in expression constructs that are intended to beplaced on the bacterial chromosome is hampered by expression of theheterologous gene from the KI plasmid intermediates. The heterologousgene is immediately preceded by a strong promoter, making that theexpression from the KI plasmid, although not intended and not required,intrinsically limits the use of the strongest promoters. In many cases,the KI plasmid has a copy number that is a multiplicity of thechromosome number in the host, making that upon integration, expressionwill be several fold lower. Therefore, chromosomal expression units willbe intrinsically weaker than what would be the highest achievable. Thisproblem is circumvented by the invention described here. In thisapproach, the heterologous genes will be positioned downstream andtranscriptionally and/or translationally coupled to a (stronglyexpressed) endogenous gene on the bacterial chromosome. This strategydoes not require the endogenous (strong) promoter to be present on theKI plasmid. Rather, upstream of the heterologous gene, the promoterless3′ end of the (strongly expressed) endogenous gene is positioned. Thistype of KI plasmid is silent and will not limit the use of strongpromoters.

The transcriptional or translational coupling of one or more exogenousgenes with one or more other genes as described herein may be achievedby means of an intergenic region active (i.e., functional, effective) ina gram-positive bacterium, preferably by means of an endogenousintergenic region of a gram-positive bacterium. Accordingly, a furtheraspect provides a recombinant nucleic acid comprising an intergenicregion active in a gram-positive bacterium, preferably an endogenousintergenic region of a gram-positive bacterium, operably linked to agene exogenous to said gram-positive bacterium. The operable linkageensures that a transcript of the intergenic region, present on a mRNAtogether with a transcript of the exogenous gene, is able to provide asite for initiation of translation of the exogenous gene, in thegram-positive bacterium. Preferably, the intergenic region may beprovided 5′ of the exogenous gene. The nucleic acid may comprise two ormore exogenous genes in polycistronic arrangement, each exogenous genepreceded by an intergenic region. The intergenic regions may be the sameor different. For example, where the intergenic regions are different,these may correspond to intergenic regions derived from different genesof the same or different species, or from the same gene of differentspecies. These nucleic acids may be useful in constructing polycistronicexpression units comprising the one or more exogenous genes, whereby oneor more other gene is transcriptionally or translationally coupled withthe one or more exogenous genes via the intergenic region. For example,these nucleic acids may be useful in constructing polycistronicexpression units as taught herein, whereby one or more endogenous genesis transcriptionally or translationally coupled with the one or moreexogenous genes via the intergenic region. Preferably the first cistronof the polycistronic expression unit will be a strongly expressedendogenous gene.

The present recombinant nucleic acids may be comprised on a replicon. Anaspect thus also relates to a replicon or vector comprising the nucleicacid as taught herein. For example, the vector may be a prokaryoticexpression vector, preferably a prokaryotic polycistronic expressionvector. The development of such plasmid expression systems may howeverbe tedious because the combination of certain replicons and strongpromoters may be unstable. Also it may be impossible to transformselected microorganisms with recombinant plasmids and stably maintainthe latter in the microorganism because of the presence of naturalplasmids, especially not if no antibiotic selection markers may beincluded in the expression plasmid, as would be the case in anapplication for therapeutic protein delivery. This issue can becircumvented by positioning the heterologous genes downstream andtranscriptionally or translationally coupled to a (strongly expressed)endogenous gene on the bacterial chromosome. As this strategy does notrequire a plasmid borne expression system, it can be used as a generalapproach to be applied for the genetic engineering of any type ofselected microflora. The only strain specific information required canbe rapidly established through state of the art technology. Highthroughput sequencing combined with proteomic analysis of the abundantlyexpressed proteins will rapidly yield the nucleotide sequence of theregions encoding the abundantly present proteins. Accordingly, mostpreferably, the vector as described herein may be configured to effecthomologous recombination in the gram-positive bacterium, such as togenerate a chromosomal integration of the exogenous gene(s).

Further provided is the use of the recombinant nucleic acid or vector asdescribed herein for polycistronic expression of the one or moreexogenous genes or for polycistronic expression of the one or moreendogenous genes and one or more exogenous genes in the gram-positivebacterium. As well disclosed is the gram-positive bacterium comprising(for example, transformed with) the recombinant nucleic acid or vectoras taught herein, whereby the gram-positive bacterium is capable ofpolycistronic expression of the one or more exogenous genes or ofpolycistronic expression of the one or more endogenous genes and one ormore exogenous genes. Further provided is a method for effectingpolycistronic expression of the one or more exogenous genes or foreffecting polycistronic expression of the one or more endogenous genesand one or more exogenous genes in a gram-positive bacterium, comprisingthe step of introducing the recombinant nucleic acid or vector as taughtherein to said gram-positive bacterium. As well provided is a method forgenerating a gram-positive bacterium capable of polycistronic expressionof the one or more exogenous genes or capable of polycistronicexpression of the one or more endogenous genes and one or more exogenousgenes, comprising the step of introducing the recombinant nucleic acidor vector as taught herein to said gram-positive bacterium.

The invention allows to express, preferably strongly (highly) express, asingle exogenous gene or a plurality of (e.g., two, three or more)distinct exogenous genes in a gram-positive bacterium. Said exogenousgene or genes may encode expression product or products such asadvantageously protein(s), polypeptide(s) and/or peptide(s). By means ofexample and not limitation, such protein(s), polypeptide(s) and/orpeptide(s) may encompass antigens (for example, for inducing immunity orimmunotolerance), allergens, non-vaccinogenic therapeutic polypeptides(cytokines, growth factors, wound healing factors, . . . ), antibodiesor functional fragments thereof (e.g., Fab fragments), fusion proteins,multimeric proteins, etc and any combination thereof.

The polycistronic organisation may render expression units as taughtherein particularly suitable for the expression of proteins comprisingtwo or more polypeptide chains (e.g., multimeric proteins, proteincomplexes). Accordingly, two or more exogenous genes as intended in thisspecification may preferably encode distinct monomers or subunits of amultimeric protein, whereby the genes are co-transcribed into apolycistronic mRNA and the individual monomers or subunits aretranslated from this mRNA. This can allow for tightly regulatedco-expression of the exogenous genes, such as to achieve balanced andoptimal assembly of the individual monomers or subunits into themultimeric protein.

A particularly advantageous illustration of this principle is theexpression of antibodies or functional fragments thereof. Hence, the twoor more exogenous genes as taught herein may preferably encode separatechains of an antibody or of a functional fragment thereof. For example,one exogenous gene may encode the light chain (V_(L)) of an antibody orof a functional fragment thereof, and another exogenous gene may encodethe heavy chain (V_(H)) of the antibody or of a functional fragmentthereof. Preferably, the functional fragment of the antibody may be Fab.In specific but non-limiting embodiments, said Fab may be binding toand/or inhibiting the biological effect of cytokines, receptors ofcytokines, chemokines or immune/inflammatory activating molecules. In apreferred embodiment the Fab may be binding to and/or inhibiting thebiological effect of TNFα, such as without limitation said Fab may becA2 anti-TNF or CDP870 anti-TNF.

The exogenous genes encoding the individual chains of the antibody or ofthe fragment thereof are thus transcriptionally or translationallycoupled for polycistronic expression in the gram-positive bacterium.Preferably, the exogenous gene encoding V_(L) or functional fragmentthereof may be transcriptionally or translationally coupled to the 3′end of the exogenous gene encoding V_(H) or functional fragment thereof.This gene organisation yields particularly effective expression andassembly of the antibody or functional fragment thereof.

The polycistronic organisation may also render expression units astaught herein particularly suitable for the co-expression of productssuch as proteins that cooperate to achieve a synergistic effect, forexample a synergistic therapeutic or prophylactic effect, for examplewhen delivered in situ by the bacterium.

Another aspect provides a gram-positive bacterium as taught herein,wherein the one or more exogenous genes encodes a product or productssuch as protein(s), polypeptide(s) or peptide(s) having a therapeutic orpreventive effect in a subject. Such bacterium is particularly providedfor use as a medicament, more particularly for use in administration ordelivery of said product or products to the subject, even moreparticularly for use in the treatment of a disease that can benefit fromthe administration or delivery of said product or products. Alsoprovided is thus a pharmaceutical composition comprising suchgram-positive bacterium.

As well provided is a method for delivering a product or products suchas protein(s), polypeptide(s) or peptide(s) to a subject comprisingadministering the gram-positive bacterium as taught herein to thesubject, wherein the one or more exogenous genes encodes said product orproducts. Preferably said product or products may have a therapeutic orpreventive effect in the subject.

Advantageously for in situ delivery of the present gram-positivebacterium to subjects, the bacterium more closely retains its endogenouscharacter by not introducing or introducing less exogenous or evenpathogenic sequences besides the sequences for the exogenous expressionproducts. Thereby, the regulatory GRAS or “Generally Recognized As Safe”status is maintained as much as possible, thus facilitating the processof acquiring clinical approval or market authorisation for the use ofthe engineered strains in humans or animals.

Further aspects and embodiments according to the invention are presentedhereafter in items (i) to (xxii).

(i) A gram-positive bacterium comprising a polycistronic expressionunit, said polycistronic expression unit consecutively comprising anendogenous gene and one or more exogenous genes transcriptionallycoupled to the 3′ end of said one or more endogenous gene.

(ii) A recombinant nucleic acid comprising a polycistronic expressionunit, said polycistronic expression unit consecutively comprising a geneendogenous to a gram-positive bacterium and one or more genes exogenousto the gram-positive bacterium transcriptionally coupled to the 3′ endof said one or more endogenous gene.

(iii) The gram-positive bacterium according to (i) or the recombinantnucleic acid according to (ii), wherein said one or more exogenous genesencodes a protein, polypeptide and/or peptide having a therapeutic orpreventive effect in a subject, or an antigen for inducing immunity orimmunotolerance, a non-vaccinogenic therapeutically active polypeptide,an antibody or a functional fragment thereof such as Fab, a fusionprotein or a multimeric protein.

(iv) The gram-positive bacterium according to (i) or the recombinantnucleic acid according to (ii), wherein the one or more exogenous genesencodes a product, such as a protein, polypeptide or peptide, whichproduct has a therapeutic or preventive effect in a subject, for use asa medicament, preferably for use in administration or delivery of saidproduct to the subject.

(v) The gram-positive bacterium according to (i), (iii) or (iv) or therecombinant nucleic acid according to (ii) to (iv), wherein said one ormore exogenous gene is the most 3′ gene of the polycistronic expressionunit.

(vi) The gram-positive bacterium according to any of (i), (iii), (iv) or(v) or the recombinant nucleic acid according to any of (ii) to (v),wherein said endogenous gene and said one or more exogenous genes aretranscriptionally controlled by a promoter endogenous to thegram-positive bacterium.

(vii) The gram-positive bacterium or the recombinant nucleic acidaccording to (vi), wherein said promoter is an essential gene promoter,a constitutive promoter, a central metabolism gene promoter, and/or ahousekeeping gene promoter.

(viii) The gram-positive bacterium or the recombinant nucleic acidaccording to (vi), wherein said promoter is a ribosomal gene promoter.

(ix) The gram-positive bacterium or the recombinant nucleic acidaccording to (vi), wherein said promoter is a glycolysis gene promoter.

(x) The gram-positive bacterium or the recombinant nucleic acidaccording to (vi), wherein said promoter is selected from the groupconsisting of the promoter of eno, usp45, gap, pyk, rpmB and rplS ofsaid gram-positive bacterium.

(xi) The gram-positive bacterium according to any one of (i) or (ii) to(x), wherein the endogenous gene is located in its native chromosomallocus in the gram-positive bacterium.

(xii) The gram-positive bacterium according to (xi), wherein theendogenous gene is transcriptionally coupled to the one or moreexogenous genes by chromosomally integrating the one or more exogenousgenes to said locus, preferably by chromosomally integrating the one ormore exogenous genes 3′ of the endogenous gene in said locus.

(xiii) The gram-positive bacterium according to any one of (i) or (iii)to (xii) or the recombinant nucleic acid according to any of (ii) to(x), wherein the endogenous gene and the one or more exogenous genes aretranscriptionally coupled by intergenic region or regions active in thegram-positive bacterium, preferably wherein the intergenic region orregions is endogenous to said gram-positive bacterium.

(xiv) The gram-positive bacterium or the recombinant nucleic acidaccording to (xiii), wherein said intergenic region is selected from thegroup consisting of intergenic regions preceding rplW, rplP, rpmD, rplB,rpsG, rpsE, rplN, rplM, rplE, and rplF.

(xv) A recombinant nucleic acid comprising an intergenic region activein a gram-positive bacterium operably linked to a gene exogenous to saidgram-positive bacterium, preferably wherein the intergenic region is anendogenous intergenic region of a gram-positive bacterium.

(xvi) The recombinant nucleic acid according to (xiv), wherein saidintergenic region is selected from the group consisting of intergenicregions preceding rplW, rplP, rpmD, rplB, rpsG, rpsE, rplN, rplM, rplE,and rplF.

(xvii) The gram-positive bacterium according to any one of (i) or (iii)to (xiv), or the recombinant nucleic acid according to any one of (ii)to (x) or (xiii) to (xvi), wherein one exogenous gene encodes the lightchain (V_(L)) of an antibody or of a functional fragment thereof, andanother exogenous gene encodes the heavy chain (V_(H)) of the antibodyor of a functional fragment thereof, more preferably wherein thefunctional fragment is Fab.

(xviii) The gram-positive bacterium or recombinant nucleic acidaccording to (xvii), wherein the exogenous gene encoding V_(L) orfunctional fragment thereof is transcriptionally coupled to the 3′ endof the exogenous gene encoding V_(H) or functional fragment thereof.

(xix) The gram-positive bacterium according to any one of (i), (iii) to(xiv), (xvii) or (xviii), or the recombinant nucleic acid according toany one of (ii) to (x), or (xiii) to (xviii), wherein the gram-positivebacterium is a lactic acid bacterium, preferably Lactococcus,Lactobacillus, or Enterococcus, more preferably Lactococcus lactis orEnterococcus faecium, or where the gram-positive bacterium is aBifidobacterium.

(xx) A pharmaceutical composition comprising the gram-positive bacteriumaccording to any one of (i), (iii) to (xiv), or (xvii) to (xix).

(xxi) The pharmaceutical composition according to (xx), wherein said oneor more exogenous genes encodes a product, such as a protein,polypeptide or peptide, which product has a therapeutic or preventiveeffect in a subject.

(xxii) A vector comprising the recombinant nucleic acid according to anyone of (ii) to (x), or (xiii) to (xix).

The above and further aspects and preferred embodiments of the inventionare described in the following sections and in the appended claims. Thesubject matter of appended claims is hereby specifically incorporated inthis specification.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Coomassie blue staining of cellular proteins of a Lactococcuslactis ssp. Cremoris strain MG1363 end-log culture. Prominent proteinbands are indicated 1 to 12.

FIG. 2: Representation of a reference, monocistronic expressionconstruct (top, sAGX0090) and polycistronic (bicistronic, dual cistron)construct according to an embodiment of the invention (bottom) wherebygene X represents an endogenous gene. Both expression constructs areintended for the expression of β-glucuronidase from the E. coli uidAgene, serving here as an exemplary exogenous gene.

FIG. 3: Relative β-glucuronidase (GUS)-activity in a reference host(monocistronic: PhIIA>>uidA, sAGX0090) and in a host comprising apolycistronic (bicistronic) construct according to an embodiment of theinvention (endogenous gene X>>rpmD>>uidA), organized as in FIG. 2. Theendogenous genes X are, in this example, usp45, enoA, rplS, rpmB, pykand gapB. In this example, the rpmD intergenic region providestranscriptional coupling of the endogenous and the exogenous gene. Theexogenous E. coli uidA gene encodes β-glucuronidase. All expressionconstructs are embedded in the bacterial chromosome. The monocistronicconstruct is present in the thyA locus, bicistronic constructs areembedded at the native position of geneX. The data show that allbicistronic constructs have b-galactosidase activity superior to themonocistronic PhIIA>>uidA construct.

FIG. 4: Quantification of human pro-insulin (ins) secretion byLactococcus lactis in a reference host (sAGX0122) and hosts according toan embodiment of the invention (sAGX0121 and sAGX0164). (A) Schematicoverview of ins expression modules. Strain sAGX0122 carries amonocistronic expression construct in which the thyA promoter drives theexpression of a secretion leader—human pro-insulin fusion (SS::ins),embedded in the Lactococcus lactis MG1363 chromosome at the thyA locus.Bicistronic expression constructs in sAGX0121 and sAGX0164 consist of atranscriptional coupling of the endogenous usp45 and enoA respectivelywith SS::ins, through the rpmD intergenic region. These constructs arelocated on the Lactococcus lactis MG1363 chromosome at the nativepositions of the usp45 and enoA genes respectively. (B) Levels ofpro-insulin detected in the supernatants of the various strains. Straincodes (sAGX0122, sAGX0121 and sAGX0164) are indicated underneath thecolumns respectively indicating human pro-insulin levels in thesupernatants of these strains. The data show that strains carrying bothbicistronic constructs have human pro-insulin levels superior to thestrain carrying the monocistronic PthyA>>ins construct.

FIG. 5: cA2 anti-TNF Fab expression in Lactococcus lactis. Genesencoding VLCL (L) and VHCH1 (H) fragments were transcriptionally coupledby rpmD, rplB, rpsG, rpsE and rplN intergenic regions. Constructs weremade in which either L or H are positioned as the first gene of thebicistronic construct. All anti-TNF expression constructs were plasmidborne and placed under the control of the PthyA promoter. Anti-TNFactivity was measured in the supernatants of the various strains. Thedata show that there is higher anti-TNF activity in all constructs whereH is the first gene of the bicistronic construct.

FIG. 6: CDP870 anti-TNF Fab expression in Lactococcus lactis. (A) CDP870light and heavy chain fusions to usp45 secretion leader encodingsequences (SS::CDP870 VLCL and SS::CDP870 VHCH1) were inserted as asecond and third cistron downstream from usp45 (sAGX0219, sAGX0220) inthe Lactococcus lactis MG1363 chromosome. In sAGX0219 and sAGX0220, rpmDwas used to couple SS::CD870 genes to usp45. To avoid geneticinstability, light and heavy chain genes were coupled through theintergenic region preceding rplN. In sAGX0219, the light chain geneprecedes the heavy chain gene, while in sAGX0220, the heavy chain geneprecedes the light chain gene. (B) Quantification of anti-human TNFactivity in crude culture supernatants. Both heavy chain and lightchains were highly expressed by the dual cistron constructs, leading tohigh levels of functional CDP870 anti-TNF Fab. CDP870 anti-TNFexpression substantially increased when the heavy chain was positionedbefore the light chain.

FIG. 7: Quantification of human trefoil factor-1 (hTFF1) secretion byLactococcus lactis in a reference host (sAGX0085) and a host accordingto an embodiment of the invention (sAGX0276). (A) Schematic overview ofhTFF1 expression modules. Strain sAGX0085 carries a monocistronicexpression construct in which the PhIIA promoter drives the expressionof a secretion leader—hTFF1 fusion (SS::hTFF1), embedded in theLactococcus lactis MG1363 chromosome at the thyA locus. The bicistronicexpression construct in sAGX0276 consist of a transcriptional couplingof gapB with SS::hTFF1, through the rpmD intergenic region. Thisconstruct is located on the Lactococcus lactis MG1363 chromosome at thenative positions of the gapB gene. (B) Levels of hTFF1 detected in thesupernatants of the various strains. Strain codes (sAGX0085 andsAGX0276) are indicated underneath the columns respectively indicatinghuman hTFF1 levels in the supernatants of these strains. The data showthat sAGX0276, carrying the bicistronic construct produces hTFF1 levelssuperior to sAGX0085 which holds the monocistronic construct.

FIG. 8: Coomassie blue staining of cellular proteins of a Enterococcusfaecium strain LMG 15709 end-log culture. Prominent protein bands areindicated 1 to 12.

FIG. 9: Representation of polycistronic (bicistronic, dual cistron)constructs according to an embodiment of the invention whereby gene Xrepresents an endogenous gene. Expression constructs are intended forthe expression of β-glucuronidase from the E. coli uidA gene, servinghere as an exemplary exogenous gene. Gap and eno are representative“fist” endogenous genes.

FIG. 10: Relative β-glucuronidase (GUS)-activity in a reference host(monocistronic: PhIIA>>uidA, sAGX0090) and in a host comprising apolycistronic (bicistronic) construct according to an embodiment of theinvention (endogenous gene X>>rpmD>>uidA), organized as in FIG. 9. Theendogenous genes X are, in this example, gapB and eno. In this example,the rpmD intergenic region provides transcriptional coupling of theendogenous and the exogenous gene. The exogenous E. coli uidA geneencodes β-glucuronidase. All expression constructs are embedded in thebacterial chromosome. The monocistronic construct is present in the thyAlocus, bicistronic constructs are embedded at the native position ofgeneX. The data show that all bicistronic constructs haveβ-galactosidase activity superior to the monocistronic PhIIA>>uidAconstruct.

FIG. 11: Quantification of human interleukin-10 (hIL-10) secretion byEnterococcus faecium in a reference host (sAGX0270) and a host accordingto an embodiment of the invention (sAGX0279). (A) Schematic overview ofhIL-10 expression modules. Bicistronic expression construct in sAGX0279consists of a transcriptional coupling of the endogenous gap withSS::hIL10, through the rpmD intergenic region. (B) Levels of hIL-10detected in the supernatants of the various strains.

FIG. 12: Quantification of human interleukin-27 (hIL-27) secretion byEnterococcus faecium in a reference host (sAGX0270) and a host accordingto an embodiment of the invention (sAGX0317). (A) Schematic overview ofhIL-27 expression modules. Bicistronic expression construct in sAGX0317consists of a transcriptional coupling of the endogenous gap withSS::hIL27, through the rpmD intergenic region. (B) Levels of hIL-27detected in the supernatants of the various strains.

FIG. 13: CDP870 anti-TNF Fab expression in Enterococcus faecium. (A)CDP870 light and heavy chain fusions to usp45 secretion leader encodingsequences (SS::CDP870 VHCH1 and SS::CDP870 VLCL) were inserted as asecond and third cistron downstream from gap (sAGX0278). To avoidgenetic instability, light and heavy chain genes were coupled throughthe intergenic region preceding rpmD from Lactococcus lactis (LL),whereas rpmD from Enterococcus faecium (EF) was used to couple gap andheavy chain genes. (B) Quantification of anti-human TNF activity incrude culture supernatants. Both heavy chain and light chains werehighly expressed by the dual cistron constructs, leading to high levelsof functional CDP870 anti-TNF Fab.

FIG. 14: Effect of anti-hTNF producing L. lactis bacteria (sAGX0220) onhTNF-induced toxicity and inflammatory cytokine production inA20^(IEC-KO) mice. (a) A20^(IEC-KO) mice (n=5 per group) were pretreatedwith vehicle, sAGX0220 or MG1363 1 hour before injection with 2 μg (leftpanel) and 6 μg (right panel) of recombinant hTNF and body temperaturewas followed in time. One group of A20^(IEC-KO) mice was injected withRemicade prior to injection with 6 μg hTNF. (b) MCP-1 levels in ileum,proximal colon and serum 5 h after injection with 2 μg of hTNF. (c) KCand IL-6 levels in ileal homogenates 5 h after injection with 2 μg hTNF.(d) MCP-1 levels in ileum, proximal colon and serum 5 h after injectionwith 6 μg of hTNF. (e) KC and IL-6 levels in ileal homogenates 5 h afterinjection with 6 μg of hTNF. Error bars represent SEM. *, p<0.05.

FIG. 15: CDP870 production in strains according to an embodiment of theinvention. (A) CDP870 heavy chain and light chain integrated in theusp45 locus, the enoA locus or the gapB locus. (B) Western blot analysisindicating CDP870 expression in different strains according to anembodiment of the invention. (C) and (D) ELISA analysis indicatingCDP870 expression in different strains according to an embodiment of theinvention. (E) TNF neutralizing activity of different strains accordingto an embodiment of the invention.

FIG. 16: Survival of Tg1278 mice with induced TNBS colitis aftertreatment with a strain according to an embodiment of the invention(anti-hTNF-secreting L. lactis strain sAGX0309) in comparison with micetreated with a wild type L. lactis strain and mice treated with Cimzia.

FIG. 17: Body weight evolution of Tg1278 mice with induced TNBS colitisafter treatment with a strain according to an embodiment of theinvention (anti-hTNF-secreting L. lactis strain sAGX0309) in comparisonwith mice treated with a wild type L. lactis strain and mice treatedwith Cimzia. Top panel: absolute body weight (g); bottom panel: bodyweight relative to starting body weight (%).

FIG. 18: Histological score of colon tissue of Tg1278 mice with inducedTNBS colitis after treatment with a strain according to an embodiment ofthe invention (anti-hTNF-secreting L. lactis strain sAGX0309) incomparison with mice treated with a wild type L. lactis strain and micetreated with Cimzia. Mean values are indicated above bars. Survival rateis indicated per group.

FIG. 19: Proinflammatory cytokine secretion in Tg1278 mice with inducedTNBS colitis after treatment with a strain according to an embodiment ofthe invention (anti-hTNF-secreting L. lactis strain sAGX0309) incomparison with healthy mice, mice treated with a wild type L. lactisstrain and mice treated with Cimzia. (A), (B), and (C) represent mIL6,mKC, and mMCP1 levels in pg/mg in the distal colon, respectively.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonymous with “including”, “includes” or “containing”, “contains”,and are inclusive or open-ended and do not exclude additional,non-recited members, elements or method steps. It will be appreciatedthat the terms “comprising”, “comprises” and “comprised of” as usedherein comprise the terms “consisting of”, “consists” and “consists of”,as well as the terms “consisting essentially of”, “consists essentially”and “consists essentially of”.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The term “about” or “approximately” as used herein when referring to ameasurable value such as a parameter, an amount, a temporal duration,and the like, is meant to encompass variations of +/−20% or less,preferably +/−10% or less, more preferably +/−5% or less, and still morepreferably +/−1% or less of and from the specified value, insofar suchvariations are appropriate to perform in the disclosed invention. It isto be understood that the value to which the modifier “about” or“approximately” refers is itself also specifically, and preferably,disclosed.

Whereas the terms “one or more” or “at least one”, such as one or moreor at least one member(s) of a group of members, is clear per se, bymeans of further exemplification, the term encompasses inter alia areference to any one of said members, or to any two or more of saidmembers, such as, e.g., any ≥3, ≥4, ≥5, ≥6 or ≥7 etc. of said members,and up to all said members.

All references cited in the present specification are herebyincorporated by reference in their entirety. In particular, theteachings of all references herein specifically referred to areincorporated by reference.

Unless otherwise defined, all terms used in disclosing the invention,including technical and scientific terms, have the meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. By means of further guidance, term definitions are included tobetter appreciate the teaching of the present invention.

In the following passages, different aspects of the invention aredefined in more detail. Each aspect so defined may be combined with anyother aspect or aspects unless clearly indicated to the contrary. Inparticular, any feature indicated as being preferred or advantageous maybe combined with any other feature or features indicated as beingpreferred or advantageous.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, appearances of the phrases“in one embodiment” or “in an embodiment” in various places throughoutthis specification are not necessarily all referring to the sameembodiment, but may. Furthermore, the particular features, structures orcharacteristics may be combined in any suitable manner, as would beapparent to a person skilled in the art from this disclosure, in one ormore embodiments. Furthermore, while some embodiments described hereininclude some but not other features included in other embodiments,combinations of features of different embodiments are meant to be withinthe scope of the invention, and form different embodiments, as would beunderstood by those in the art. For example, in the appended claims, anyof the claimed embodiments can be used in any combination.

In the following detailed description of the invention, reference ismade to the accompanying drawings that form a part hereof, and in whichare shown by way of illustration only of specific embodiments in whichthe invention may be practiced. It is to be understood that otherembodiments may be utilised and structural or logical changes may bemade without departing from the scope of the present invention. Thefollowing detailed description, therefore, is not to be taken in alimiting sense, and the scope of the present invention is defined by theappended claims.

Standard reference works setting forth the general principles ofrecombinant DNA technology include Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989; Current Protocols inMolecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates) (“Ausubel etal. 1992”); Innis et al., PCR Protocols: A Guide to Methods andApplications, Academic Press: San Diego, 1990. General principles ofmicrobiology are set forth, for example, in Davis, B. D. et al.,Microbiology, 3rd edition, Harper & Row, publishers, Philadelphia, Pa.(1980).

As noted an aspect of the invention relates to a gram-positive bacteriumcomprising an endogenous gene to which one or more exogenous genes aretranscriptionally or translationally coupled. Preferably, the one ormore exogenous genes are transcriptionally or translationally coupleddownstream (i.e. at the 3′ end) of the endogenous gene. A related aspectprovides a gram-positive bacterium comprising a polycistronic expressionunit, said polycistronic expression unit comprising an endogenous geneand one or more exogenous genes. Preferably, the polycistronicexpression unit consecutively comprises one or more endogenous genes andone or more exogenous genes. A further aspect provides a recombinantnucleic acid comprising a polycistronic expression unit comprising agene endogenous to a gram-positive bacterium to which one or more genesexogenous to the gram-positive bacterium are transcriptionally ortranslationally coupled. Preferably, the one or more exogenous genes aretranscriptionally or translationally coupled downstream (i.e. at the 3′end) of the endogenous gene.

Preferably, the one or more exogenous gene(s) is (are) the most 3′ genesof the polycistronic expression unit, i.e. the one or more exogenousgene(s) is (are) the last or most downstream gene(s) of thepolycistronic expression unit. For instance if the endogenous gene ismonocistronic, the one or more exogenous gene is located after ordownstream (i.e. at the 3′ end) of—and transcriptionally coupledwith—the open reading frame of the gene. Likewise, if the endogenousgene is itself polycistronic, such as (part of) an operon, the one ormore exogenous gene is located after or downstream (i.e. at the 3′ end)of the last (i.e. most downstream or most 3′) endogenous gene of theendogenous polycistronic gene.

Most preferably, the endogenous gene as referred to herein throughoutthe description is monocistronic. The endogenous gene preferably thusdoes not form part of an endogenous operon.

Preferably, the expression of the polycistronic expression unit asdescribed herein is effected by a promoter which may be or may exhibitone or more of the following characteristics: constitutive promoters,central metabolism gene promoters, essential gene promoters, strongpromoters, housekeeping gene promoters, ribosomal gene promoters,glycolysis gene promoters. Most preferably, the promoter is aconstitutive promoter.

As used herein, the term “gram-positive bacterium” has its commonmeaning known in the art. By means of further guidance, a gram-positivebacterium can be identified by Gram staining as retaining crystal violetstain.

In a preferred embodiment, the gram-positive bacterium according to theinvention is non-pathogenic in the sense that it does not cause harm ordoes not lead to deleterious effects when administered to an intendedsubject.

Preferably, the gram-positive bacterium according to the invention is alactic acid bacterium (LAB), including, but not limited to the generaLactococcus, Lactobacillus, Leuconostoc, Pediococcus, Streptococcus,Aerococcus, Carnobacterium, Enterococcus, Oenococcus,Sporolactobacillus, Tetragenococcus, Vagococcus, and Weisella. Morepreferably, the LAB is a Lactococcus species, such as, but not limitedto Lactococcus lactis, Lactococcus garvieae, Lactococcus piscium,Lactococcus plantarum and Lactococcus raffinolactis, and any subspeciesand strains thereof. Most preferably, the Lactococcus species isLactococcus lactis, and any subspecies and strain thereof, such aswithout limitation Lactococcus lactis ssp. cremoris, Lactococcus lactisssp. hordniae, Lactococcus lactis ssp. lactis, Lactococcus lactis ssp.bv. diacetylactis. In further preferred embodiments of the invention theLactococcus lactis is Lactococcus lactis ssp. cremoris or Lactococcuslactis ssp. lactis, more preferably Lactococcus lactis ssp. cremoris,and encompasses any strains thereof, such as, e.g., Lactococcus lactisssp. cremoris SK11, Lactococcus lactis ssp. cremoris MG1363, orLactococcus lactis ssp lactis IL1403. In another preferred embodiment,the LAB is an Enterococcus sp., preferably Enterococcus faecalis,Enterococcus faecium and any subspecies and strains thereof, such as,without limitation Enterococcus faecium strain LMG15709.

In another preferred embodiment, the gram-positive bacterium accordingto the invention is Bifidobacterium.

Bifidobacterium is a genus of Gram-positive, non-motile, often branchedanaerobic bacteria. Bifidobacteria as used herein may include B.adolescentis, B. angulatum, B. animalis, B. asteroides, B. bifidum, B.boum, B. breve, B. catenulatum, B. choerinum, B. coryneforme, B.cuniculi, B. denticolens, B. dentium, B. gallicum, B. gallinarum, B.indicum, B. infantis, B. inopinatum, B. lactis, B. longum, B. magnum, B.merycicum, B. minimum, B. pseudocatenulatum, B. pseudolongum, B.pullorum, B. ruminantium, B. saeculare, B. subtile, B. suis, B.thermacidophilum, B. thermophilum. Preferably, the Bifidobacterium is B.adolescentis, B. bifidum, B. breve, B. infantis, B. longum. It is to beunderstood that all subspecies and strains of Bifidobacteria are alsoincluded.

As used herein, the term “consecutively” in the context of endogenousand exogenous genes refers to the 5′ to 3′ order of the respective genesin a polynucleic acid, vector or chromosome. For example, apolycistronic expression unit consecutively comprising one or moreendogenous genes and one or more exogenous genes relates to a unit inwhich the one or more endogenous genes are positioned upstream of theone or more exogenous genes. Hence the one or more exogenous genes arepositioned after the 3′ end of the one or more endogenous genes. It isto be understood that the consecutive coupling or ordering as describedherein does not necessarily imply a direct coupling of the endogenousand exogenous gene. Additional sequences may be present between theendogenous and exogenous gene. As an example, an intergenic region asdefined further herein may be present between (i.e. downstream or 3′ ofthe endogenous gene and upstream or 5′ of the exogenous gene) theconsecutive endogenous and exogenous genes. As used herein, the terms“endogenous gene”, “endogenous promoter”, “endogenous intergenicregion”, “endogenous ribosome binding site” refer to respectively agene, promoter, intergenic region or ribosome binding site which arenative to a gram-positive bacterium, or can be found in nature in agram-positive bacterium. As such, the term endogenous gene, promoter,intergenic region or ribosome binding site encompasses orthologousgenes, promoters, intergenic regions, and ribosome binding sites betweendifferent genera, species, subspecies or strains of gram-positivebacteria. In particular, a gene, promoter, intergenic region or ribosomebinding site isolated from one genus, species, subspecies, or strain ofgram-positive bacteria is said to be endogenous for all other genera,species, subspecies, or strains of gram-positive bacteria, irrespectiveof possible polynucleic acid sequence differences, provided said othergenus, species, subspecies, or strain of gram-positive bacteria innature also comprises such gene, promoter, intergenic region or ribosomebinding site. Thus, such divergent but found-in-nature gene, promoter,intergenic region, or ribosome binding site sequences would beconsidered endogenous. By means of example, and without limitation, thegene encoding enolase, enoA, which is isolated from Lactococcus lactisssp. lactis is also considered endogenous in respect of Lactococcuslactis ssp. cremoris.

Preferably, however, an “endogenous” gene, promoter or intergenic regionof a given genus, species, subspecies or strain of gram-positivebacterium as intended herein may denote a gene, promoter or intergenicregion which is found in nature in, i.e., is native to or own to, thatsame genus, species, subspecies or strain of gram-positive bacterium,respectively. By means of example, and without limitation, the geneencoding enolase, enoA, which is isolated from Lactococcus lactis ssp.lactis may preferably be considered “endogenous” to Lactococcus lactisssp. lactis, but not to Lactococcus lactis ssp. cremoris.

As used herein, the term “exogenous gene” refers to a gene which is notnative to a gram-positive bacterium, or cannot be found in nature in agram-positive bacterium. The term exogenous gene is synonymous with theterm heterologous gene. The exogenous gene may be a full length gene ormay alternatively be a truncated gene or a gene fragment. By means ofexample, a exogenous gene can be derived from viruses, otherprokaryotes, such as a gram-negative bacterium, or alternatively andpreferably can be derived from eukaryotes, such as plants, animals,preferably mammals, most preferably human. Alternatively, the exogenousgene can be completely or partially synthetic or artificial, in thesense that it completely or partially does not occur in nature. Inaddition, the exogenous gene can be chimeric, in the sense that it canbe composed of sequences originating from different species or acombination of naturally occurring and synthetic or artificialsequences. Also encompassed are chimeric sequences composed ofgram-positive bacterial sequences and sequences exogenous ofgram-positive bacteria, such as for instance sequences encoding fusionproteins composed of gram-positive bacterial secretion signal peptidesand exogenous proteins.

As eukaryotic genes for the most part comprise introns beside exons, theskilled person will appreciate that according to the invention, anyreference to a exogenous gene relates to the intron-less open readingframe of such gene, i.e., the protein-coding sequence of such gene. Theterm “open reading frame” or ORF refers to a succession of codingnucleotide triplets starting with a translation initiation codon (e.g.ATG or GTG) and closing with a translation termination codon (e.g., TAA,TAG or TGA) and encoding a single polypeptide.

Prokaryotic genes, in particular genes from gram-positive bacteria donot comprise introns. Hence, the coding sequence or open reading frameof a prokaryotic gene corresponds to the succession of coding nucleotidetriplets starting with a translation initiation codon and closing with atranslation termination codon as located on the prokaryotic genome, inparticular the bacterial chromosome.

Accordingly, in an aspect, the invention relates to a gram-positivebacterium comprising an endogenous open reading frame or coding sequenceto which one or more exogenous open reading frame or coding sequencesare transcriptionally or translationally coupled.

The skilled person will understand that, whereas the term “gene” ingeneral may refer to a locatable region of genomic sequence,corresponding to a unit of inheritance, which is associated withtranscriptional and translational regulatory regions such as the pribnowbox, shine-dalgarno sequence, operators, terminators, transcribedregions, and or other functional sequence regions, any reference to theterm “gene” in the context of exogenous sequences as described hereinpreferably refers to the coding sequence or open reading frame of thatgene, unless explicitly stated to the contrary. Any reference to theterm “gene” in the context of endogenous sequences as described hereinmay refer to a locatable region of genomic sequence, corresponding to aunit of inheritance, which is associated with regulatory regions,transcribed regions, and or other functional sequence regions, butalternatively may also refer to the coding sequence or open readingframe of that gene.

As used herein, the term “translationally coupled” is synonymous with“translationally linked” or “translationally connected”. These terms inessence relate to polycistronic expression systems or units. Two or moregenes, open reading frames or coding sequences are said to betranslationally coupled when common regulatory element(s) such as inparticular a common promoter effects the transcription of said two ormore genes as one mRNA encoding said two or more genes, open readingframes or coding sequences, which can be subsequently translated intotwo or more individual polypeptide sequences. The skilled person willappreciate that bacterial operons are naturally occurring polycistronicexpression systems or units in which two or more genes aretranslationally or transcriptionally coupled. According to theinvention, transcriptional coupling underlies translational coupling.

Accordingly, in an aspect, the invention relates to a gram-positivebacterium comprising an endogenous gene to which one or more exogenousgenes, open reading frame or coding sequence are transcriptionallycoupled. Preferably, the gram-positive bacterium consecutively comprisesan endogenous gene to which one or more exogenous genes, open readingframe or coding sequence are transcriptionally coupled. As used herein,the term “transcriptionally coupled” is synonymous with“transcriptionally connected” and “transcriptionally linked”. Theseterms generally refer to polynucleic acid sequences comprising two ormore open reading frames or coding sequences which are commonlytranscribed as one mRNA, and which can be translated into two or moreindividual polypeptides.

In other aspects, the invention relates to a gram-positive bacterium orrecombinant nucleic acid comprising a polycistronic expression unit,said polycistronic expression unit comprising an endogenous gene and oneor more exogenous genes, open reading frame or coding sequence.

As used herein, the term “polycistronic expression unit” or“polycistronic expression system” refers to a unit wherein theexpression of two or more genes is regulated by common regulatorymechanisms, such as promoters, operators, and the like. The termpolycistronic expression unit as used herein is synonymous withmulticistronic expression unit. Examples of polycistronic expressionunits are without limitation bicistronic, tricistronic, tetracistronicexpression units. Any mRNA comprising two or more, such as 3, 4, 5, 6,7, 8, 9, 10, or more, open reading frames or coding regions encodingindividual expression products such as proteins, polypeptides and/orpeptides is encompassed within the term polycistronic.

In an embodiment, the translationally or transcriptionally coupled oneor more endogenous genes and one or more exogenous genes as describedherein are transcriptionally controlled by a promoter which isendogenous to a gram-positive bacterium. In another embodiment, thepolycistronic expression unit or system as described herein istranscriptionally controlled by a promoter which is endogenous to a grampositive bacterium.

By “promoter” is meant generally a region on a nucleic acid molecule,preferably DNA molecule, to which an RNA polymerase binds and initiatestranscription. A promoter is preferably, but not necessarily, positionedupstream, i.e., 5′, of the sequence the transcription of which itcontrols.

In a further embodiment, the translationally or transcriptionallycoupled one or more endogenous genes and one or more exogenous genes asdescribed herein are transcriptionally controlled by the native promoterof (one of) said one or more endogenous genes. In another embodiment,the polycistronic expression unit or system as described herein istranscriptionally controlled by the native promoter of (one of) said oneor more endogenous genes comprised in said polycistronic expressionsystem or unit. In another embodiment, the polycistronic expression unitor system as described herein is operably linked to a gram-positiveendogenous promoter.

As used herein, the term “operably linked” or “operable linkage” is alinkage in which the regulatory DNA sequences and the DNA sequencesought to be expressed are connected in such a way as to permitexpression. For example, a promoter is said to be operably linked to agene, open reading frame or coding sequence, if the linkage orconnection allows or effects transcription of said gene. In a furtherexample, a 5′ and a 3′ gene, cistron, open reading frame or codingsequence are said to be operably linked in a polycistronic expressionunit, if the linkage or connection allows or effects translation of atleast the 3′ gene.

For example, DNA sequences, such as, e.g., preferably a promoter and anopen reading frame, are said to be operably linked if the nature of thelinkage between the sequences does not (1) result in the introduction ofa frame-shift mutation, (2) interfere with the ability of the promoterto direct the transcription of the open reading frame, or (3) interferewith the ability of the open reading frame to be transcribed by thepromoter region sequence.

In an exemplary preferred embodiment, the promoter may be positionedupstream of, i.e., 5′ of, the open reading frame(s) to which it isoperably linked.

The skilled person will appreciate that the promoter may be associatedwith additional native regulatory sequences or regions, e.g. operators.The precise nature of the regulatory regions needed for expression mayvary from organism to organism, but shall in general include a promoterregion which, in prokaryotes, contains both the promoter (which directsthe initiation of RNA transcription) as well as the DNA sequences which,when transcribed into RNA, will signal the initiation of proteinsynthesis. Such regions will normally include those 5′-non-codingsequences involved with initiation of transcription and translation,such as the Pribnow-box (cf. TATA-box), Shine-Dalgarno sequence, and thelike.

In a further embodiment, the promoter is the native promoter of the 5′most, i.e., most upstream, endogenous gene in the polycistronicexpression unit.

As used herein, the term “constitutive” in the context of a promoter (orby extension relating to gene expression of the endogenous gene) refersto a promoter that allows for continual transcription of its associatedgene. In particular, transcription of the associated gene or genes undercontrol of such promoter occurs independently of any inducer or otherregulatory signal.

As used herein, the term “housekeeping gene” or “housekeeping promoter”refers to a gene or a promoter of a gene that is required for themaintenance of basic cellular function. Although some housekeeping genesare expressed at relatively constant levels, other housekeeping genesmay vary depending on external or experimental conditions. Housekeepinggenes may for instance be involved in metabolism, gene expression (suchas basal transcription machinery), signalling, but may also bestructural genes.

As used herein, “glycolysis gene” or “glycolysis promoter” refers to agene or promoter of a gene involved in the glycolytic pathway, andinclude the promoters of the genes encoding glycolytic enzymes,particularly hexokinase, phosphoglucose isomerase, phosphofructokinase,fructose bisphosphate aldolase, triosephosphate isomerase,glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase,phosphoglycerate mutase, enolase, and pyruvate kinase.

As used herein, “ribosomal gene” or “ribosomal promoter” refers to agene or promoter of a ribosomal gene, including genes encoding ribosomalproteins as well as genes transcribed into ribosomal RNA. Preferably, itmay refer to a gene or promoter of a ribosomal protein.

As used herein, “central metabolism gene” or “central metabolismpromoter” or alternatively “basic metabolism gene” or “basic metabolismpromoter” refers to a gene or promoter of a gene involved in criticalmetabolic pathways, and includes genes involved in glycolysis,pentose-phosphate pathway, and tricarboxylic acid (TCA) cycle.

As used herein, the term “essential” in the context of a gene (or byextension relating to the promoter of such gene) relates to a gene theabsence of the native expression product of which is detrimental, suchas in particular lethal, for the host or alternatively alters, inhibitsor prevents, normal physiology or function, such as in particularpropagation or growth. It is to be understood that, as used herein, theterm “essential” in the context of a gene or promoter of a gene relatesto constitutively essential, as opposed to conditionally essential. Forinstance, the genes of the lactose operon, such as thebeta-galactosidase gene, in several gram-positive bacteria, inparticular lactic acid bacteria such as Lactococcus sp. may be essentialwhen the bacteria are cultivated in a medium containing lactose as themain or sole carbon source, these genes are not essential when thebacteria are cultivated in a medium containing alternative carbonsources. These genes are therefore only conditionally essential, but notconstitutively essential, as intended herein.

In a preferred embodiment, the endogenous promoter and/or the endogenousgene as described herein is selected from the group comprising orconsisting of gram-positive bacterial promoters and/or genescorresponding to the following Lactococcus promoters and/or genes, moreparticularly Lactococcus lactis ssp. cremoris strain MG1363 promotersand/or genes: 1) DNA-directed RNA polymerase, beta′ subunit/160 kDsubunit (rpoC), 2) DNA-directed RNA polymerase, beta subunit/140 kDsubunit (rpb2 or rpoB), 3) DNA-binding ferritin-like protein (oxidativedamage protectant) (dps), 4) pyruvate kinase (pyk), 5) glutamyl- andglutaminyl-tRNA synthetases (glnS or gltX), 6) enolase (eno), 7)glutamine synthetase (glnA) 8) HTH-type transcriptional regulator(glnR), 9) Xaa-His dipeptidase (argE or pepV), 10) F0F1-type ATPsynthase beta subunit (ATP synthase F1 beta subunit) (atpD), 11)3-phosphoglycerate kinase (pgk), 12) glyceraldehyde-3-phosphatedehydrogenase/erythrose-4-phosphate dehydrogenase (gapA or gapB), 13)acetate kinase (ackA), 14) 3-oxoacyl-(acyl-carrier-protein) synthase(fabB or fabF), 15) 3-oxoacyl-(acyl-carrier-protein) reductase (fabG),16) DNA-directed RNA polymerase, alpha subunit/40 kD subunit (rpoA), 17)Xaa-Pro aminopeptidase (pepP), 18) fructose/tagatose bisphosphatealdolase (tbp or fbaA), 19) ribosomal protein S4 (rpsD), 20) superoxidedismutase (sodA), 21) ribosomal protein S12 (rpsL) and ribosomal proteinS7 (rpsG), 22) ribosomal protein L18 (rplR) and ribosomal protein S5(rpsE) and ribosomal protein L30/L7E (rpmD), 23) S-ribosylhomocysteinelyase (luxS), 24) ribosomal protein L19 (rplS), 25) ribosomal proteinS11 (rpsK), 26) ribosomal protein L10 (rplJ), 27) ribosomal proteinL7/L12 (rplL), 28) bacterial nucleoid DNA-binding protein/DNA bindingprotein HU (hup or hllA), 29) 50S ribosomal protein L28 (rpmB), 30)phosphotransferase system cellobiose-specific component IIB (lace orptcB), 31) F0F1-type ATP synthase alpha subunit (atpA), 32) ABC-typesugar transport system (ATPase component) (malK or msmK), 33) acetoindehydrogenase complex E1 component alpha subunit (acoA or pdhA), 34)cell division protein (diflVA or ftsA), 35) UDP-galactopyranose mutase(glf), 36) glutamyl aminopeptidase (frvX or pepA), 37) predicteddehydrogenase related protein (mviM or llmg_0272), 38) ribosomal proteinS2 (rpsB), 39) translation initiation factor 3 (IF-3) (infC), 40)ribosomal protein L4 (rplD) and ribosomal protein L23 (rplW) andribosomal protein L2 (rplB), 41) EMAP domain (ydjD), 42) transcriptionelongation factor (greA), 43) protease subunit of ATP-dependent Clpprotease (clpP), 44) ribosomal protein L15 (rplO), 45) ribosomal proteinL11 (rplK), 46) ribosomal protein S8 (rpsH), 47) ribosomal protein L21(rplU), 48) ribosomal protein S13 (rpsM), 49) ribosomal protein S19(rpsS) and ribosomal protein L22 (rplU or rplV) and ribosomal proteinL16 (rplP) and ribosomal protein L14 (rplN), 50) ribosomal protein S10(rpsJ), 51) co-chaperonin GroES (Hsp10) (cpn10), 52) ribosomal proteinL24 (rplX), 53) hypothetical protein LACR_0137 (duf965), and 54)secreted 45 kDa protein (usp45). Preferably, the endogenous promoterand/or endogenous gene is selected from the group comprising orconsisting of enoA, usp45, gapB, pyk, rpmB, and rplS. These promotersand their sequences are disclosed for example in WO 2008/08411incorporated by reference herein, e.g., in Table 1 and FIG. 1A-Hthereof. In an embodiment, the invention relates to a gram positivebacterium or a recombinant nucleic acid as described herein, wherein theendogenous gene and the one or more exogenous genes aretranscriptionally controlled by a promoter endogenous to thegram-positive bacterium, preferably by an endogenous promoter selectedfrom the group consisting of the promoter of eno, usp45, gap, pyk, rpmBand rplS of said gram-positive bacterium. In a further embodiment, theendogenous gene is located in its native chromosomal locus in thegram-positive bacterium.

In a preferred embodiment, said one or more exogenous genes, openreading frames or coding sequences are translationally ortranscriptionally coupled to the 3′ end of said one or more endogenousgenes, open reading frame or coding sequence. Accordingly, in anembodiment, the invention provides for a gram-positive bacteriumcomprising a polycistronic expression unit, wherein said polycistronicexpression unit comprises one or more 5′ endogenous gene and one or more3′ exogenous gene. Preferably, the 5′ most gene of the polycistronicexpression unit is an endogenous gene. By means of example, and withoutlimitation, the polycistronic expression unit may comprise or consistessentially of from 5′ end to 3′ end an endogenous gene followed by oneor more endogenous genes, followed by one or more exogenous genes.Alternatively, and without limitation, the polycistronic expression unitmay comprise or consist essentially of from 5′ end to 3′ end anendogenous gene followed by one or more exogenous genes. Alternatively,the polycistronic expression unit may comprise or consist essentially offrom 5′ end to 3′ end an endogenous gene followed by one or moreexogenous genes, followed by one or more endogenous genes.

The translationally coupled or transcriptionally coupled one or moreendogenous genes and one or more exogenous genes, or the polycistronicexpression unit or system, as described herein may be comprised in areplicon which allows maintenance and/or propagation and expression ofthe endogenous and exogenous genes in the gram-positive bacteriaaccording to the invention as described herein.

In an embodiment, the translationally coupled or transcriptionallycoupled one or more endogenous genes and one or more exogenous genes,optionally including the (endogenous) promoter as described elsewhere inthis specification, or the polycistronic expression unit or system, asdescribed herein may be comprised in a vector, preferably an expressionvector allowing expression in gram-positive bacteria. Accordingly, theinvention also relates to a vector comprising the recombinant nucleicacid as described herein.

As used herein, “vector” refers to a nucleic acid molecule, typicallyDNA, to which nucleic acid fragments may be inserted and cloned, i.e.,propagated. Hence, a vector will typically contain one or more uniquerestriction sites, and may be capable of autonomous replication in adefined host or vehicle organism such that the cloned sequence isreproducible. Vectors may include, without limitation, plasmids,phagemids, bacteriophages, bacteriophage-derived vectors, PAC, BAC,linear nucleic acids, e.g., linear DNA, etc., as appropriate (see, e.g.,Sambrook et al., 1989; Ausubel 1992).

Factors of importance in selecting a particular vector, e.g., a plasmid,include inter alia: the ease with which recipient cells that contain thevector may be recognized and selected from those recipient cells whichdo not contain the vector; the number of copies of the vector which aredesired in a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species. Preferredprokaryotic vectors include plasmids such as those capable ofreplication in E. coli (such as, for example, pBR322, ColE1, pSC101,pUC19, etc.). Such plasmids are describe in, e.g., Sambrook et al.,1989; Ausubel 1992. Particularly preferred vectors may be those able toreplicate in E. coli (or other Gram negative bacteria) as well as inanother host cell of interest, such as in a Gram positive bacterium, alactic acid bacterium, preferably Lactococcus, more preferablyLactococcus lactis (see, e.g., Kok et al. Appl. Environ. Microbiol.,1984, vol. 48(4), 726-31). Other preferred vectors may be those able toreplicate and/or shuttle between one or more Gram positive bacteria butnot in Gram negative bacteria. In a preferred embodiment, the vector ispT1NX as described by Steidler et al. Appl. Environ. Microbiol., 1995,vol. 61(4), 1627-1629, which is specifically incorporated by referenceherein.

In another embodiment, the translationally coupled or transcriptionallycoupled one or more endogenous genes and one or more exogenous genes, orthe polycistronic expression unit or system, as described herein areintegrated in the gram-positive bacterial genome or chromosome. Methodsfor obtaining recombinant gram-positive bacteria and random as well ashomologous recombination are well-known in the art, as well as vectorsfor effecting recombination. By means of further guidance, such methodsand vectors are for instance disclosed in Steidler et al. (2003, NatureBiotechnology, 21:785-789), Law et al. (1995, J Bacteriol, 177(24):7011-7018), Leenhouts et al. (1998, Methods in Cell Science, 20:35-50)and WO 2004/046346, which are incorporated in its entirety by reference.Preferably, the polycistronic expression unit as described herein isgenerated or introduced by site-directed integration of the requisitesequences in the bacterial chromosome by homologous recombination.

In an embodiment, a recombination vector comprises an endogenouspromoter as described elsewhere in this specification, and optionallyadditional regulatory sequences, as well as a polycistronic expressionunit as described herein. Preferably, the endogenous promoter and thepolycistronic expression unit are operably linked. Homologousrecombination can be effected at a predetermined locus. Such system ishighly modular and allows for individual selection and combination ofpromoter, regulatory sequences, endogenous gene, and exogenous gene, aswell as the choice of insertion site.

In another embodiment, the gram-positive bacterium according to theinvention comprises an endogenous promoter as described elsewhere inthis specification at its native locus, i.e., in its native genomiccontext on the bacterial chromosome, to which a polycistronic expressionunit comprising one or more endogenous genes, open reading frame orcoding sequence and one or more exogenous genes, open reading frame orcoding sequence are operably linked. The operable linkage can beeffected through homologous recombination between the locus comprisingthe promoter and a recombination vector comprising the polycistronicexpression unit, flanked by sequences configured to effect saidhomologous recombination. Accordingly, in an embodiment, the inventionrelates to a gram-positive bacterium as described herein, wherein theendogenous gene is transcriptionally coupled to the one or moreexogenous genes by chromosomally integrating the one or more exogenousgenes to said locus, preferably by chromosomally integrating the one ormore exogenous genes 3′ of the endogenous gene in said locus.

Vector design can be chosen such that merely the open reading frame orcoding sequences of the endogenous and/or exogenous genes are integratedinto the intended chromosomal locus. In this case, the regulatorysequences beside the promoter per se which effect transcription and/ortranslation, e.g. operators, transcription initiation site,shine-dalgarno sequence, terminator sequence, etc. are provided for bythe native genomic locus of the promoter. Alternatively, such sequencesmay be provided on the recombination vector comprising the polycistronicexpression unit. In the latter case, depending on the needs, the nativeregulatory sequences associated with the endogenous promoter may beremoved during homologous recombination. The systems described here aremodular in respect of individual selection of endogenous gene, exogenousgene and possibly the regulatory sequences, but predetermine theinsertion site at the endogenous locus of the selected promoter.

In a further embodiment, the gram-positive bacterium according to theinvention comprises an endogenous promoter as well as one or moreendogenous genes, both as described elsewhere in this specification, atits (their) native locus, i.e. in its (their) native genomic context onthe bacterial chromosome, to which one or more exogenous genes, openreading frame or coding sequence are operably linked, such as to effectpolycistronic expression of the one or more endogenous genes and the oneor more exogenous genes. In this system, the endogenous promoter and oneor more endogenous genes, as well as the regulatory sequences effectingtranscription and translation of said one or more endogenous genes ispresent in their native locus. Such system maximally preserves thenative character of the gram-positive bacterium.

A polycistronic expression unit comprises at least two genes, openreading frames or coding sequences. In order to initiate translation ofall genes, each of these genes generally is associated with sequenceseffecting ribosome binding, i.e. ribosome binding sites. In prokaryotes,ribosome binding sites are denoted Shine-Dalgarno (SD) sequences, whichhave the general consensus sequence 5′-AGGAGG-3′. The SD sequence onaverage is located about 8 base pairs upstream (i.e. 5′ of) thetranslation initiation codon or start codon. Depending on the distance(in amount of nucleotides) between the stop codon of the 5′ gene and thestart codon of the 3′ gene, the SD sequences can be typicallypositioned 1) in an intergenic region between both genes, if thedistance is at least the size of the SD sequence; 2) in an intergenicregion between both genes, but overlapping with the stop codon of the 5′gene, in case of a smaller distance between 5′ and 3′ gene; or 3) 5′ tothe stop codon of the 5′ gene, if for instance the stop codon of the 5′gene and the start codon of the 3′ gene are very close or overlap.

In an embodiment, the invention relates to a gram-positive bacterium ora recombinant nucleic acid as described herein, further comprising oneor more polynucleic acid sequences comprising a ribosome binding siteconfigured to effect translation of the one or more exogenous genes. Inanother embodiment, the invention relates to a gram-positive bacteriumor a recombinant nucleic acid as described herein, further comprisingone or more ribosome binding site configured to effect translation ofthe one or more exogenous genes. In a further embodiment, the inventionrelates to a gram-positive bacterium or a recombinant nucleic acid asdescribed herein wherein said one or more endogenous genes and said oneor more exogenous genes are transcriptionally or translationally coupledby means of a ribosome binding site. In yet another embodiment, theinvention relates to a gram-positive bacterium or a recombinant nucleicacid, comprising a polycistronic expression unit as described herein,wherein any 5′ gene is coupled to a 3′ gene by a polynucleic acidsequence comprising or consisting (essentially) of a ribosome bindingsite. In a preferred embodiment, said ribosome binding site isendogenous to a gram-positive bacterium. In a further preferredembodiment, said ribosome binding site is comprised in an intergenicregion, preferably an operon intergenic region.

In another embodiment, the invention relates to a gram-positivebacterium or a recombinant nucleic acid as described herein, furthercomprising one or more polynucleic acid sequences comprising anintergenic region configured to effect translation of the one or moreexogenous genes. In a further embodiment, the invention relates to agram-positive bacterium or a recombinant nucleic acid as describedherein, further comprising one or intergenic region configured to effecttranslation of the one or more exogenous genes. In a further embodiment,the invention relates to a gram-positive bacterium or a recombinantnucleic acid as described herein wherein said one or more endogenousgenes and said one or more exogenous genes are transcriptionally ortranslationally coupled by means of an intergenic region. In yet anotherembodiment, the invention relates to a gram-positive bacterium or arecombinant nucleic acid, comprising a polycistronic expression unit asdescribed herein, wherein any 5′ gene is coupled to a 3′ gene by apolynucleic acid sequence comprising or consisting of an intergenicregion. In a preferred embodiment, said intergenic region is endogenousto a gram-positive bacterium. In a further preferred embodiment, saidintergenic region is an operon intergenic region.

As used herein, the term “intergenic region” is synonymous with“intergenic linker” or “intergenic spacer”. An intergenic region isdefined as a polynucleic acid sequence between adjacent (i.e., locatedon the same polynucleic acid sequence) genes, open reading frames,cistrons or coding sequences. By extension, the intergenic region caninclude the stop codon of the 5′ gene and/or the start codon of the 3′gene which are linked by said intergenic region. As defined herein, theterm intergenic region specifically relates to intergenic regionsbetween adjacent genes in a polycistronic expression unit. For example,an intergenic region as defined herein can be found between adjacentgenes in an operon. Accordingly, in an embodiment, the intergenic regionas defined herein is an operon intergenic region.

In an embodiment, the intergenic region, linker or spacer is selectedfrom the group of intergenic regions comprising or consisting ofintergenic regions preceding, i.e. 5′ to, more particularly immediately5′ to, rplW, rplP, rpmD, rplB, rpsG, rpsE or rplN of a gram-positivebacterium. In an embodiment, said gram positive bacterium is a lacticacid bacterium, preferably a Lactococcus species, more preferablyLactococcus lactis, and any subspecies or strain thereof. In anembodiment, said intergenic region encompasses the start codon of rplW,rplP, rpmD, rplB, rpsG, rpsE or rplN and/or the stop codon of thepreceding, i.e. 5′, gene. In a preferred embodiment, the inventionrelates to a gram-positive bacterium or a recombinant nucleic acid asdescribed herein, wherein the endogenous gene and the one or moreexogenous genes are transcriptionally coupled by intergenic region orregions active in the gram-positive bacterium, preferably wherein theintergenic region or regions is endogenous to said gram-positivebacterium, more preferably wherein the endogenous intergenic region isselected from the group consisting of intergenic regions preceding rplW,rplP, rpmD, rplB, rpsG, rpsE, rplN, rplM, rplE, and rplF.

The skilled person will appreciate that if the intergenic regionencompasses a 5′ stop codon and/or a 3′ start codon, these respectivecodons preferably are not present in the genes which are linked by saidintergenic regions, in order to avoid double start and/or stop codons,which may affect correct translation initiation and/or termination.Methods for identifying intergenic regions are known in the art. Bymeans of further guidance, intergenic regions can for instance beidentified based on prediction of operons, and associated promoters andopen reading frames, for which ample software is known and available inthe art.

In a further embodiment, said intergenic region sequence is selectedfrom the group comprising, consisting essentially of or consisting ofany of SEQ ID NOs: 1 to 7:

SEQ ID NO: 1 TAATG SEQ ID NO: 2 TAATCCATG SEQ ID NO: 3 TAAGGAGGAAAAAATGSEQ ID NO: 4 TAATAGAGGAGGAAAATCGTG SEQ ID NO: 5 TAAGAAGGGAGATAAGTAAGAATGSEQ ID NO: 6 TAAGGAAAGGGGTAATTAAACATG SEQ ID NO: 7TAAGCAAAACTAGGAGGAATATAGCATG.

In a further embodiment, said intergenic region sequence is selectedfrom the group comprising, consisting essentially of or consisting ofsequences displaying one mismatch or a deletion or insertion of onenucleotide vs. SEQ ID NO: 1 or SEQ ID NO: 2, sequences displaying one,two or three mismatches, or a deletion or insertion of one, two or threenucleotides vs. SEQ ID NO: 3 or SEQ ID NO: 4, and sequences displayingone, two, three or four mismatches or a deletion or insertion of one,two, three or four nucleotides vs. SEQ ID NO: 5, SEQ ID NO: 6 or SEQ IDNO: 7.

SEQ ID NOs: 1 to 7 all comprise a 5′ stop codon and a 3′ start codon.SEQ ID NOs: 1 to 7 correspond to the intergenic regions preceding,respectively, rplW, rplP, rpmD, rplB, rpsG, rpsE and rplN of Lactococcuslactis ssp. cremoris strain MG1363 (Genbank accession numberAM406671.1). These sequences are among other identical to thecorresponding sequences of Lactococcus lactis ssp. lactis strain CV56(Genbank accession number CP002365.1), Lactococcus lactis ssp. cremorisstrain NZ9000 (Genbank accession number CP002094.1), Lactococcus lactisssp. lactis strain KF147 (Genbank accession number CP001834.1),Lactococcus lactis ssp. lactis strain IL1403 (Genbank accession numberAE005176.1), and Lactococcus lactis ssp. cremoris strain SK11 (Genbankaccession number CP000425.1).

In another embodiment, the intergenic region, linker or spacer isselected from the group of intergenic regions comprising or consistingof intergenic regions preceding, i.e. 5′ to, more particularlyimmediately 5′ to, rplP, rpmD, rplM, rpsE, rplE, or rplF of agram-positive bacterium. In an embodiment, said gram positive bacteriumis a lactic acid bacterium, preferably an Enterococcus species, morepreferably Enterococcus faecium, and any subspecies or strain thereof.In an embodiment, said intergenic region encompasses the start codon ofrplP, rpmD, rplM, rpsE, rplE, or rplF and/or the stop codon of thepreceding, i.e. 5′, gene. The skilled person will appreciate that if theintergenic region encompasses a 5′ stop codon and/or a 3′ start codon,these respective codons preferably are not present in the genes whichare linked by said intergenic regions, in order to avoid double startand/or stop codons, which may affect correct translation initiationand/or termination. Methods for identifying intergenic regions are knownin the art. By means of further guidance, intergenic regions can forinstance be identified based on prediction of operons, and associatedpromoters and open reading frames, for which ample software is known andavailable in the art.

In a further embodiment, said intergenic region sequence is selectedfrom the group comprising, consisting essentially of or consisting ofany of SEQ ID NOs: 8 to 13:

SEQ ID NO: 8 TAATC SEQ ID NO: 9 TAAGGAGGACAACAATA SEQ ID NO: 10TAATAGGAGGGAATTTCA SEQ ID NO: 11 TTAGAAGAAGGAGGAATACCATTC SEQ ID NO: 12TAAAAGTTTAAGGAAGGAGGGTCTTACTGA SEQ ID NO: 13TAATCAAGTAGAATCTACAAGGAGGTGTCTTTAA

In a further embodiment, said intergenic region sequence is selectedfrom the group comprising, consisting essentially of or consisting ofsequences displaying one mismatch or a deletion or insertion of onenucleotide vs. SEQ ID NO: 8, sequences displaying one, two or threemismatches, or a deletion or insertion of one, two or three nucleotidesvs. SEQ ID NO: 9 or SEQ ID NO: 10 and sequences displaying one, two,three or four mismatches or a deletion or insertion of one, two, threeor four nucleotides vs. SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13.SEQ ID NOs: 8 to 13 correspond to the intergenic regions preceding,respectively, rplP, rpmD, rplM, rpsE, rplE, and rplF of Enterococcusfaecium strain LMG15709

In an embodiment, the intergenic regions as described herein, excludingany preceding stop codon and excluding any subsequent start codon maycomprise or consist of more than 1 nucleotide, such as 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore nucleotides, preferably more than 5 nucleotides, even morepreferably 10 or more nucleotides. In another embodiment, the intergenicregion may comprise 1 to 50 nucleotides, such as 1 to 40, 1 to 30, 1 to25, 1 to 20, 1 to 15, or 1 to 10, preferably 5 to 50, 5 to 40, 5 to 30,5 to 25, 5 to 20, 5 to 15, or 5 to 10 nucleotides, even more preferably10 to 50, 10 to 40, 10 to 30, 10 to 25, 10 to 20, or 10 to 15nucleotides.

Particularly preferred embodiments of gram-positive bacteria comprisinga polycistronic expression unit as described herein are depicted inTables 1 and 2, wherein said gram-positive bacterium comprises anendogenous promoter, 3′ of which the endogenous gene coupled to theintergenic region as depicted in Tables 1 and 2, 3′ of which one or moreexogenous genes, open reading frame or coding sequence coupled to theintergenic region. In a preferred embodiment, each gene depicted inTables 1 and 2 is transcriptionally controlled by its native promoter,and optionally regulatory sequences. In another preferred embodiment,said polycistronic expression unit is integrated in the bacterialchromosome. In a further preferred embodiment, said endogenous promoterand/or endogenous gene are present at their native locus on thebacterial genome or chromosome. Preferably, the start and stop codons,if present, replace the start and stop codons of said exogenous gene andsaid endogenous gene, respectively.

TABLE 1 Exemplary polycistronic expression unit may comprise or consistessentially of endogenous promoter >> endogenous gene >> intergenicregion >> exogenous gene, wherein the endogenous gene and intergenicregion are selected from the combinations below. endogenous geneintergenic region eno rplW eno rplP eno rpmD eno rplB eno rpsG eno rpsEeno rplN eno rplM eno rplE eno rplF usp45 rplW usp45 rplP usp45 rpmDusp45 rplB usp45 rpsG usp45 rpsE usp45 rplN usp45 rplM usp45 rplE usp45rplF gap rplW gap rplP gap rpmD gap rplB gap rpsG gap rpsE gap rplN gaprplM gap rplE gap rplF pyk rplW pyk rplP pyk rpmD pyk rplB pyk rpsG pykrpsE pyk rplN pyk rplM pyk rplE pyk rplF rpmB rplW rpmB rplP rpmB rpmDrpmB rplB rpmB rpsG rpmB rpsE rpmB rplN rpmB rplM rpmB rplE rpmB rplFrplS rplW rplS rplP rplS rpmD rplS rplB rplS rpsG rplS rpsE rplS rplNrplS rplM rplS rplE rplS rplF

Preferably, intergenic regions rplW, rplB, rpsG, and rplN originate froma Lactococcus species, subspecies or strain, preferably Lactococcuslactis. Preferably, intergenic regions rplP, rplM, and rplE originatefrom an Enterococcus species, subspecies or strain, preferablyEnterococcus faecalis or Enterococcus Faecium. Preferably, intergenicregions rplP, rpmD, and rpsE originate from a Lactococcus species,subspecies or strain, preferably Lactococcus lactis or from anEnterococcus species, subspecies or strain, preferably Enterococcusfaecalis or Enterococcus Faecium.

For example but without limitation, where the polycistronic expressionunit comprises two exogenous genes, the structure represented asendogenous promoter>>endogenous gene>>intergenic region>>exogenousgene>>intergenic region>>exogenous gene may be as follows:usp45>>usp45>>rpmD>>exogenous gene 1>>rplN>>exogenous gene 2;enoA>>enoA>>rpmD>>exogenous gene 1>>rplN>>exogenous gene 2;gapB>>gapB>>rpmD>>exogenous gene 1>>rplN>>exogenous gene 2. For example,such arrangement may be particularly suited for the expression of heavyand light chains of antibodies (preferably in that order), such asanti-TNFα antibodies as taught herein.

TABLE 2 Exemplary polycistronic expression unit may comprise or consistessentially of endogenous promoter >> endogenous gene >> intergenicregion >> exogenous gene, wherein the endogenous gene and intergenicregion are selected from the combinations below. endogenous geneintergenic region eno SEQ ID NO: 1 eno SEQ ID NO: 2 eno SEQ ID NO: 3 enoSEQ ID NO: 4 eno SEQ ID NO: 5 eno SEQ ID NO: 6 eno SEQ ID NO: 7 eno SEQID NO: 8 eno SEQ ID NO: 9 eno SEQ ID NO: 10 eno SEQ ID NO: 11 eno SEQ IDNO: 12 eno SEQ ID NO: 13 usp45 SEQ ID NO: 1 usp45 SEQ ID NO: 2 usp45 SEQID NO: 3 usp45 SEQ ID NO: 4 usp45 SEQ ID NO: 5 usp45 SEQ ID NO: 6 usp45SEQ ID NO: 7 usp45 SEQ ID NO: 8 usp45 SEQ ID NO: 9 usp45 SEQ ID NO: 10usp45 SEQ ID NO: 11 usp45 SEQ ID NO: 12 usp45 SEQ ID NO: 13 gap SEQ IDNO: 1 gap SEQ ID NO: 2 gap SEQ ID NO: 3 gap SEQ ID NO: 4 gap SEQ ID NO:5 gap SEQ ID NO: 6 gap SEQ ID NO: 7 gap SEQ ID NO: 8 gap SEQ ID NO: 9gap SEQ ID NO: 10 gap SEQ ID NO: 11 gap SEQ ID NO: 12 gap SEQ ID NO: 13pyk SEQ ID NO: 1 pyk SEQ ID NO: 2 pyk SEQ ID NO: 3 pyk SEQ ID NO: 4 pykSEQ ID NO: 5 pyk SEQ ID NO: 6 pyk SEQ ID NO: 7 pyk SEQ ID NO: 8 pyk SEQID NO: 9 pyk SEQ ID NO: 10 pyk SEQ ID NO: 11 pyk SEQ ID NO: 12 pyk SEQID NO: 13 rpmB SEQ ID NO: 1 rpmB SEQ ID NO: 2 rpmB SEQ ID NO: 3 rpmB SEQID NO: 4 rpmB SEQ ID NO: 5 rpmB SEQ ID NO: 6 rpmB SEQ ID NO: 7 rpmB SEQID NO: 8 rpmB SEQ ID NO: 9 rpmB SEQ ID NO: 10 rpmB SEQ ID NO: 11 rpmBSEQ ID NO: 12 rpmB SEQ ID NO: 13 rplS SEQ ID NO: 1 rplS SEQ ID NO: 2rplS SEQ ID NO: 3 rplS SEQ ID NO: 4 rplS SEQ ID NO: 5 rplS SEQ ID NO: 6rplS SEQ ID NO: 7 rplS SEQ ID NO: 8 rplS SEQ ID NO: 9 rplS SEQ ID NO: 10rplS SEQ ID NO: 11 rplS SEQ ID NO: 12 rplS SEQ ID NO: 13

Preferably, the gram-positive bacterium having a polycistronicexpression unit comprising any of SEQ ID NOs: 1 to 7 is a Lactococcusspecies, subspecies or strain, preferably Lactococcus lactis.Preferably, the gram-positive bacterium having a polycistronicexpression unit comprising any of SEQ ID NOs: 8 to 13 is an Enterococcusspecies, subspecies or strain, preferably Enterococcus faecalis orEnterococcus Faecium.

The skilled person will appreciate that the exogenous genes, openreading frames or coding sequences according to the invention can becoupled to additional sequences, which additional sequences effect aparticular purpose. For instance, in order to increase secretion of theexogenous gene, the gene may be coupled to a nucleic acid sequenceencoding a secretion signal peptide. In a particularly preferredembodiment, the exogenous gene, open reading frame or coding sequenceaccording to the invention is coupled at its 5′ end to the polynucleicacid sequence encoding the Usp45 secretion signal, preferablyoriginating from a Lactococcus species, more preferably Lactococcuslactis and subspecies and strains thereof.

Typically, a secretion signal sequence represents an about 16 to about35 amino acid segment, usually containing hydrophobic amino acids thatbecome embedded in the lipid bilayer membrane, and thereby allow for thesecretion of an accompanying protein or peptide sequence from the hostcell, and which usually is cleaved from that protein or peptide.Preferably, the secretion signal sequence may be so-active in a hostcell intended for use with the nucleic acid comprising the said signalsequence.

Secretion signal sequences active in suitable host cells are known inthe art; exemplary Lactococcus signal sequences include those of usp45(see, U.S. Pat. No. 5,559,007) and others, see, e.g., Perez-Martinez etal. Mol. Gen. Genet., 1992, vol. 234, 401-11; Sibakov et al., Appl.Environ. Microbiol., 1991, vol. 57(2), 341-8. Preferably, the signalsequence is located between the promoter sequence and the ORF, i.e. thesignal sequence is located 3′ from the promoter sequence and precedesthe ORF of the polypeptide of interest. In a preferred embodiment, thesignal sequence encodes the amino acid sequenceMKKKIISAILMSTVILSAAAPLSGVYA (usp45). Alternatively, a mutated usp45signal sequence (usp45N) may be used which results in furthercontrollable production and secretion of the polypeptide of interest. Inparticular, the mutant comprises an asparagine (N) at position 4 insteadof a lysine (K), or a K4N mutation. In a preferred embodiment, thesignal sequence encodes the amino acid sequence MKKNIISAILMSTVILSAAAPLSGVYADTN.

The invention also relates to a polynucleic acid sequence comprising apolycistronic expression unit according to the invention as describedherein. In particular, in an aspect, the invention relates to apolynucleic acid sequence comprising a polycistronic expression unitaccording to the invention as described herein, wherein saidpolycistronic unit comprises one or more gene endogenous to agram-positive bacterium and one or more gene, open reading frame orcoding sequence exogenous to a gram-positive bacterium, wherein the oneor more endogenous genes and the one or more exogenous genes aretranslationally or transcriptionally coupled in a way as describedherein. Preferably the one or more endogenous genes is coupled to the 5′end of the one or more exogenous genes. Preferably, the one or moreendogenous genes and the one or more exogenous genes are connected by anintergenic region as described herein, preferably an intergenic regionpreceding rplW, rplP, rpmD, rplB, rpsG, rpsE, and rplN as describedherein elsewhere or an intergenic region corresponding to any of SEQ IDNOs: 1 to 7 or an intergenic region preceding rplP, rpmD, rplM, rpsE,rplE, or rplF as described herein elsewhere or an intergenic regioncorresponding to any of SEQ ID NOs: 8 to 13 or related sequences asdescribed above. In an embodiment, the polynucleic acid sequence furthercomprises a promoter, preferably a promoter endogenous of agram-positive bacterium. In another embodiment, the polynucleic acidsequence further comprises regulatory sequences, e.g. operator,terminator and the like. In a preferred embodiment, the promoter is thenative promoter of the endogenous gene.

In a further aspect, the invention relates to a replicon comprising thepolynucleic acid sequence as described herein. Preferably, said repliconis a vector, as described herein elsewhere. In an embodiment, saidvector is suitable for prokaryotic expression. In another embodiment,said vector is suitable for homologous recombination in a gram-positivebacterium.

In another aspect, the invention relates to a polynucleic acid sequencecomprising a ribosome binding site of a gram positive bacterium and agene, open reading frame or coding sequence exogenous to said bacterium,wherein the ribosome binding site is configured to effect translation ofthe exogenous gene, open reading frame or coding sequence. In anembodiment, the polynucleic acid sequence comprises a ribosome bindingsite of a gram positive bacterium and a gene, open reading frame orcoding sequence exogenous to said bacterium, wherein the ribosomebinding site is connected at the 5′ end of the exogenous gene, openreading frame or coding sequence.

In another aspect, the invention relates to a polynucleic acid sequencecomprising an intergenic region, preferably an operon intergenic region,of a gram positive bacterium and a gene, open reading frame or codingsequence exogenous to said bacterium, wherein the intergenic region isconfigured to effect translation of the exogenous gene, open readingframe or coding sequence. In an embodiment, the polynucleic acidsequence comprises an intergenic region, preferably an operon intergenicregion, of a gram positive bacterium and a gene, open reading frame orcoding sequence exogenous to said bacterium, wherein the intergenicregion is connected at the 5′ end of the exogenous gene, open readingframe or coding sequence. Preferably, the intergenic region is anintergenic region preceding rplW, rplP, rpmD, rplB, rpsG, rpsE, or rplNas described herein elsewhere or an intergenic region corresponding toany of SEQ ID NOs: 1 to 7 or an intergenic region preceding rplP, rpmD,rplM, rpsE, rplE, or rplF as described herein elsewhere or an intergenicregion corresponding to any of SEQ ID NOs: 8 to 13 or related sequencesas described above.

In a further aspect, the invention relates to a polycistronic expressionvector comprising the intergenic region preceding rplW, rplP, rpmD,rplB, rpsG, rpsE, or rplN as described herein elsewhere or an intergenicregion corresponding to any of SEQ ID NOs: 1 to 7 or an intergenicregion preceding rplP, rpmD, rplM, rpsE, rplE, or rplF as describedherein elsewhere or an intergenic region corresponding to any of SEQ IDNOs: 8 to 13 or related sequences as described above. In an embodiment,said vector is suitable for cloning a gene, open reading frame or codingsequence at the 3′ end of said intergenic region, preferably a genewhich is exogenous to a gram-positive bacterium. In an embodiment, saidvector is suitable for being replicated in a gram-positive bacterium. Ina further embodiment, said vector is suitable for effecting homologousrecombination in a gram-positive bacterium, in particular forchromosomal integration of said intergenic region and a gene, openreading frame or coding sequence at the 3′ end of said intergenicregion. In an embodiment, said vector further comprises one or morepromoter, preferably a gram-positive bacterial promoter. In a furtherembodiment, said vector further comprises regulatory sequences, e.g.operator, terminator and the like. In yet another embodiment, saidvector further comprises one or more selection markers, such asantibiotic resistance genes.

In another aspect, the invention relates to a method for exogenous geneexpression in a gram-positive bacterium, comprising the step oftransforming said gram-positive bacterium with the vector comprising anexogenous gene, open reading frame or coding sequence, optionallyfurther comprising an (endogenous) promoter as described herein.

In a further aspect, the invention relates to the use of a polynucleicacid sequence comprising an intergenic region of a gram-positivebacterium as described herein for polycistronic expression of one ormore gene, open reading frame or coding sequence exogenous to saidgram-positive bacterium. In an embodiment, the invention relates to theuse of a polynucleic acid sequence comprising an intergenic region of agram-positive bacterium as described herein for polycistronic expressionof one or more genes, open reading frames or coding sequences exogenousto said gram-positive bacterium and one or more gene, open reading frameor coding sequences endogenous to said gram-positive bacterium. In anembodiment, said one or more genes exogenous to said gram-positivebacterium is coupled to the 3′ end of said endogenous gene. Preferably,the intergenic region is an intergenic region preceding rplW, rplP,rpmD, rplB, rpsG, rpsE or rplN as described herein elsewhere or anintergenic region corresponding to any of SEQ ID NOs: 1 to 7 or anintergenic region preceding rplP, rpmD, rplM, rpsE, rplE, or rplF asdescribed herein elsewhere or an intergenic region corresponding to anyof SEQ ID NOs: 8 to 13 or related sequences as described above.

In another aspect, the invention relates to a method for expressing ofone or more exogenous protein in a gram-positive bacterium, comprisingthe step of introducing a polynucleic acid sequence encoding said one ormore exogenous protein or a vector as described herein in saidgram-positive bacterium such as to be transcribed in a polycistronicmRNA.

In yet another aspect, the invention relates to a method for generatinga gram-positive bacterium capable of expressing one or more exogenousproteins, comprising the step of introducing a polynucleic acid sequenceencoding one or more exogenous protein or a vector as described hereinin said gram-positive bacterium such as to be transcribed in apolycistronic mRNA.

According to the invention, the one or more exogenous genes, openreading frame of coding sequence can be of any kind or origin. In anembodiment, the one or more exogenous genes encodes a protein,polypeptide and/or peptide, preferably a protein, polypeptide and/orpeptide having a therapeutic or preventive effect in a subject, orpreferably an antigen such as an antigen for inducing immunity orimmunotolerance, a non-vaccinogenic therapeutically active polypeptide,an antibody or a functional fragment thereof such as Fab, a fusionprotein or a multimeric protein. In a preferred embodiment, the one ormore exogenous genes encodes an antibody or a functional antibodyfragment. As used herein, the term “functional” refers to an antibodyfragment, which can still exert its intended function, i.e. antigenbinding. The term antibody, as used here, includes, but is not limitedto conventional antibodies, chimeric antibodies, dAb, bispecificantibody, trispecific antibody, multispecific antibody, bivalentantibody, trivalent antibody, multivalent antibody, VHH, nanobody, Fab,Fab′, F(ab′)₂ scFv, Fv, dAb, Fd, diabody, triabody, single chainantibody, single domain antibody, single antibody variable domain.

In the present context, the term “antibody” is used to describe animmunoglobulin whether natural or partly or wholly engineered. Asantibodies can be modified in a number of ways, the term “antibody”should be construed as covering any specific binding molecule orsubstance having a binding domain with the required binding specificityfor the other member of the pair of molecules, i.e. the target molecule,as defined supra. Thus, this term covers antibody fragments,derivatives, functional equivalents and homologues of antibodies, aswell as single chain antibodies, bifunctional antibodies, bivalentantibodies, VHH, nanobodies, Fab, Fab′, F(ab′)₂, scFv, Fv, dAb, Fd,diabodies, triabodies and camelid antibodies, including any polypeptidecomprising an immunoglobulin binding domain, whether natural or whollyor partially engineered. Chimeric molecules comprising an immunoglobulinbinding domain, or equivalent, fused to another polypeptide aretherefore included. The term also covers any polypeptide or proteinhaving a binding domain which is, or is homologous to, an antibodybinding domain, e.g. antibody mimics. Examples of antibodies are theimmunoglobulin isotypes and their isotypic subclasses, including IgG(IgG1, IgG2a, IgG2b, IgG3, IgG4), IgA, IgD, IgM and IgE. The person inthe art will thus appreciate that the present invention also relates toantibody fragments, comprising an antigen binding domain such as VHH,nanobodies Fab, scFv, Fv, dAb, Fd, diabodies and triabodies. In anembodiment, the invention relates to a gram-positive bacterium or arecombinant nucleic acid as described herein, wherein one exogenous geneencodes the light chain (V_(L)) of an antibody or of a functionalfragment thereof, and another exogenous gene encodes the heavy chain(V_(H)) of the antibody or of a functional fragment thereof, morepreferably wherein the functional fragment is Fab. In an embodiment, theexogenous gene encoding V_(L) or functional fragment thereof istranscriptionally coupled to the 3′ end of the exogenous gene encodingV_(H) or functional fragment thereof.

In an embodiment, the antibody as described herein at least partially orfully blocks, inhibits, or neutralises a biological activity of a targetmolecule, such as a cytokine or chemokine. As used herein, theexpression “neutralises” or “neutralisation” means the inhibition of orreduction in a biological activity of a cytokine as measured in vivo orin vitro, by methods known in the art, such as, for instance, asdetailed in the examples. In particular, the inhibition or reduction maybe measured by determining the colitic score or by determining thetarget molecule in a tissue or blood sample. As used herein, theexpression “neutralises” or “neutralisation” means the inhibition of orreduction in a biological activity of a cytokine as measured in vivo orin vitro, by at least 10% or more, preferably by at least 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% and even more preferably by 100%.

Preferably, said binding molecules are binding to and inhibiting thebiological effect of cytokines chosen from the list of IL-1β, IL-2,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12 (or its subunits IL-12p35 andIL12p40), IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23 (or itssubunit IL-23p19), IL-27, IL-32 (and its splice variants), IFN (α, β, γ)and TNFα. Preferably, said binding molecules are soluble cytokinereceptors such as gp130, or are binding to the receptors of saidcytokines, for example IL-2R (CD25, CD122, CD132), IL-12R (beta1,beta2), IL15R, IL-17R, IL-23R or IL-6R, without triggering aninflammatory signal. Preferably, said binding molecules are neutralizingchemokines chosen from the list of MIF, MIP-1α, MCP-1, RANTES andEotaxin. Preferably, said binding molecules are solving the blockade ofimmune activation via binding to costimulatory molecules from the listof CD3/CD28, HVEM, B7.1/B7.2, CD40/CD40L(CD154), ICOS/ICOSL, OX40/X40L,CD27/CD27L(CD70), CD30/CD30L(CD153) and 41BB/41BBL. Preferably, saidbinding molecules are solving the blockade of inflammation via bindingto adhesion molecules from the list I-CAM1, α4 integrin and α4β7integrin. Preferably, said binding molecules have a costimulatory andagonistic effect on CD3, CTLA4 and/or PD1. Preferably, said bindingmolecules are neutralizing T-cells or B-cell activity by targeting CD25,CD20, CD52, CD95, BAFF, APRIL and/or IgE. Preferably, said bindingmolecules are solving the blockade of inflammation via binding toenzymes from the MMP family. Preferably, said binding molecules assertan anti-angiogenic effect, such as neutralizing αvβ3/α5β1 and IL-8activity. In a further preferred embodiment said binding molecule iscapable of neutralizing the biological effect of TNFα, IL-12, IFNγ,IL-23 or IL-17. Preferably, said binding molecule is chosen from thegroup consisting of

-   -   an anti-TNFα antibody, anti-TNFα antibody fragment, anti-TNFα        single antibody variable domain, soluble TNF receptor or        dominant negative variant of TNFα;    -   anti-IL-12 antibody, anti-IL-12 antibody fragment, anti-IL-12        single antibody variable domain, soluble IL-12 receptor,        dominant negative variant of IL-12 or IL-12 dAb;    -   anti-IL-12p35 antibody, anti-IL-12p35 antibody fragment,        anti-IL-12p35 single antibody variable domain, soluble IL-12p35        receptor, dominant negative variant of IL-12p35 or IL-12p35 dAb;    -   anti-IL-12p40 antibody, anti-IL-12p40 antibody fragment,        anti-IL-12p40 single antibody variable domain, soluble IL-12p40        receptor, dominant negative variant of IL-12p40 or IL-12p40 dAb;    -   anti-IL-23 antibody, anti-IL-23 antibody fragment, anti-IL-23        single antibody variable domain, soluble IL-23 receptor,        dominant negative variant of IL-23 or IL-23 dAb;    -   anti-IL-23p19 antibody, anti-IL-23p19 antibody fragment,        anti-IL-23p19 single antibody variable domain, soluble IL-23p19        receptor, dominant negative variant of IL-23p19 or IL-23p19 dAb;    -   an anti-IFNγ antibody, anti-IFNγ antibody fragment, anti-IFNγ        single antibody variable domain, soluble IFNγ receptor or        dominant negative variant of IFNγ;    -   anti-IL-17 antibody, anti-IL-17 antibody fragment, anti-IL-17        single antibody variable domain, soluble IL-17 receptor,        dominant negative variant of IL-17 or IL-17 dAb; and    -   anti-MCP-1 antibody, anti-MCP-1 antibody fragment, anti-MCP-1        single antibody variable domain, soluble IL-17 receptor,        dominant negative variant of MCP-1 or MCP-1 dAb.

In a preferred embodiment, said antibody is a Fab fragment (fragmentantigen-binding). Fab fragments are well known in the art. By means offurther guidance, a Fab fragment is a region on an antibody that bindsto antigens. It is composed of one constant and one variable domain ofeach of the heavy and the light chain.

In an embodiment, the Fab is cA2 anti-TNF Fab (of which thepolynucleotide and polypeptide sequences of the variable domain of theheavy chain and the light chain are disclosed in U.S. Pat. No. 6,790,444as SEQ ID NO: 4 and 5 (heavy chain) and SEQ ID NO: 2 and 3 (lightchain), respectively) or CDP870 anti-TNF Fab (of which thepolynucleotide and polypeptide sequences of the heavy chain and thelight chain are disclosed in WO 01/94585 as SEQ ID NO: 114 and 115(heavy chain) and SEQ ID NO: 112 and 113 (light chain), respectively).

The skilled person will appreciate that antibodies, as are functionalantibody fragments, and in particular Fab fragments, are composed ofdifferent individual polypeptides which may be covalently linked bydisulphide bridges. In particular, the heavy chain and the light chainare encoded by separate individual coding sequences.

Accordingly, the coding regions of the heavy and light chains may eachbe comprised in a polycistronic expression unit as described herein.Polynucleic acid sequences encoding heavy and light chains may beincorporated in different polycistronic expression units. Preferably,polynucleic acid sequences encoding heavy and light chains areincorporated in the same polycistronic expression unit. Accordingly, inan embodiment, the invention relates to a gram-positive bacterium asdescribed herein, comprising one or more endogenous genes, one or morepolynucleic acid sequence encoding an antibody heavy chain, or afragment, preferably a functional fragment thereof, and one or morepolynucleic acid sequence encoding an antibody light chain, or afragment, preferably a functional fragment thereof, which aretranslationally or transcriptionally coupled. In another embodiment, theinvention relates to a gram-positive bacterium comprising apolycistronic expression unit, wherein said polycistronic expressionunit comprises one or more endogenous genes, one or more polynucleicacid sequence encoding an antibody heavy chain, or a fragment,preferably a functional fragment thereof, and one or more polynucleicacid sequence encoding an antibody light chain, or a fragment,preferably a functional fragment thereof. In yet another embodiment, thepolynucleic acid sequence encoding a light chain is transcriptionally ortranslationally coupled to 3′ end of the polynucleic acid sequenceencoding the heavy chain. Advantageously, such coupling furtherincreases the expression of both heavy and light chain.

The invention also relates to the use of the gram-positive bacteriaaccording to the invention as described herein for therapy. Theinvention further relates to a pharmaceutical composition comprising thegram-positive bacterium according to the invention as described herein.

Accordingly, in an aspect, the invention relates to the gram-positivebacterium or a pharmaceutical composition comprising the gram-positivebacterium according to the invention as described herein for use as amedicament. In another aspect, the invention relates to thegram-positive bacterium or a pharmaceutical composition comprising thegram-positive bacterium according to the invention as described hereinfor use in therapy or treatment. In a further aspect, the inventionrelates to the use of the gram-positive bacterium or a pharmaceuticalcomposition comprising the gram-positive bacterium according to theinvention as described herein for the manufacture of a medicament. Inyet another aspect, the invention relates to a method of treatment,comprising administering the gram-positive bacterium or a pharmaceuticalcomposition comprising the gram-positive bacterium according to theinvention as described herein. In an embodiment, the invention relatesto a gram-positive bacterium or a pharmaceutical composition comprisinga gram-positive bacterium as described herein, wherein the one or moreexogenous genes encodes a product, such as a protein, polypeptide orpeptide, which product has a therapeutic or preventive effect in asubject, preferably for use as a medicament, preferably for use inadministration or delivery of said product to the subject.

In a related aspect, the invention provides a method for delivery of apolypeptide encoded by the one or more exogenous genes, open readingframe, or coding sequence comprised in the gram-positive bacterium ofthe invention to human or animal in need thereof, comprisingadministering to said human or animal a therapeutically effective amountof gram-positive bacteria according to the invention as describedherein. The animal may preferably be a mammal, such as, e.g., domesticanimals, farm animals, zoo animals, sport animals, pet and experimentalanimals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses,cattle, cows; primates such as apes, monkeys, orang-utans, andchimpanzees; canids such as dogs and wolves; felids such as cats, lions,and tigers; equids such as horses, donkeys, and zebras; food animalssuch as cows, pigs, and sheep; ungulates such as deer and giraffes;rodents such as mice, rats, hamsters and guinea pigs; and so on.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder. A “human or animal in need oftreatment” includes ones that would benefit from treatment of a givencondition.

The term “therapeutically effective amount” refers to an amount of atherapeutic substance or composition effective to treat a disease ordisorder in a subject, e.g., human or animal, i.e., to obtain a desiredlocal or systemic effect and performance. By means of example, atherapeutically effective amount of bacteria may comprise at least 1bacterium, or at least 10 bacteria, or at least 10² bacteria, or atleast 10³ bacteria, or at least 10⁴ bacteria, or at least 10⁵ bacteria,or at least 10⁶ bacteria, or at least 10⁷ bacteria, or at least 10⁸bacteria, or at least 10⁹, or at least 10¹⁰, or at least 10¹¹, or atleast 10¹², or at least 10¹³, or at least 10¹⁴, or at least 10¹⁵, ormore gram-positive bacteria, e.g., in a single or repeated dose.

The gram-positive bacteria of the present invention may be administeredalone or in combination with one or more active compounds. The lattercan be administered before, after or simultaneously with theadministration of the gram-positive bacteria.

A number of prior art disclosures on the delivery of antigens and/ortherapeutically active polypeptides exist, and it shall be appreciatedthat such disclosures may be further advantageously modified with thegram-positive bacteria of the present invention. By means of example andnot limitation, bacterial delivery of interleukins in particular IL-10for treating colitis (e.g. WO 00/23471), IL-27 for modulating aninflammatory response (WO 2004/069177), delivery of antigens as vaccines(e.g. WO 97/14806), delivery of GLP-2 and related analogs may be used totreat short bowel disease, Crohn's disease, osteoporosis and as adjuvanttherapy during cancer chemotherapy, etc. Furthermore, bacterial deliveryof trefoil peptides may be used to treat diseases of the alimentarycanal (e.g. WO 01/02570). In particular, the use of trefoil proteins orpeptides for treatment of disorders of and damage to the alimentarycanal, including the mouth, oesophagus, stomach, and large and smallintestine, as well as for the protection and treatment of tissues thatlie outside the alimentary canal are described in WO 97/38712 and WO92/14837. These proteins can be used either to treat lesions in theseareas or to inhibit the formation of lesions. These lesions can becaused by: radiation therapy or chemotherapy for the treatment ofcancer, any other drug including alcohol which damages the alimentarycanal, accidental exposure to radiation or to a caustic substance,infection, a digestive disorder including but not limited to oralmucositis, intestinal mucositis, esophagitis, proctitis, non-ulcerdyspepsia, gastritis, peptic or duodenal ulcer, gastric cancer, coloncancer, MALT lymphoma, Menetier's syndrome, gastro-oesophageal refluxdisease, Crohn's disease, ulcerative colitis and acute colitis ofchemical, bacterial or obscure origin. Trefoil peptides are particularlyuseful to treat acute colitis, oral mucositis, intestinal mucositis,esophagitis, proctitis. Further therapeutic applications are envisionedusing the promoters and host cells of the invention.

Further non-limiting examples of the types of diseases treatable inhumans or animals by delivery of therapeutic polypeptides according tothe invention include, but are not limited to, e.g., inflammatory boweldiseases including Crohn's disease and ulcerative colitis (treatablewith, e.g., IL-Ira, IL-10, IL-27 or trefoil peptides); autoimmunediseases, including but not limited to psoriasis, rheumatoid arthritis,lupus erythematosus (treatable with, e.g., IL-Ira, IL-27, IL-10 or therelevant auto-antigen); allergic diseases including but not limited toasthma, food allergies, (treatable with the relevant allergen); celiacdisease (treatable with gluten allergens); neurological disordersincluding, but not limited to Alzheimer's disease, Parkinson's diseaseand amyotrophic lateral sclerosis (treatable with, e.g., brain devatedneurotropic factor and ciliary neurotropic factor); cancer (treatablewith, e.g., IL-1, colony stimulating factors or interferon-W);osteoporosis (treatable with, e.g., transforming growth factor f3);diabetes (treatable with, e.g., insulin); cardiovascular disease(treatable with, e.g., tissue plasminogen activator); atherosclerosis(treatable with, e.g., cytokines and cytokine antagonists); hemophilia(treatable with, e.g., clotting factors); degenerative liver disease(treatable with, e.g., hepatocyte growth factor or interferon a);pulmonary diseases such as cystic fibrosis (treatable with, e.g., alphaantitrypsin); obesity; pathogen infections, e.g., viral or bacterialinfections (treatable with any number of the above-mentionedcompositions or antigens); etc.

The gram-positive bacteria according to the invention can also be usedto treat infectious diseases. In an embodiment, passive immunizationagainst Clostridium associated disease, preferably Clostridium dificileassociated disease (CDAD), with toxin-neutralizing antibodies locallyproduced and secreted via the gram-positive bacterium according to theinvention can be obtained. Preferably, said gram positive bacterium is aLactococcus sp., more preferably Lactococcus lactis or a subspecies or astrain thereof.

CDAD is mediated by two exotoxins, toxin A (enterotoxin; see forinstance Genbank NC_009089.1, region: 795843 . . . 803975 for DNAsequence or YP_001087137.1 for protein sequence) and toxin B (cytotoxin;see for instance Genbank NC_009089.1, region: 787393 . . . 794493 forDNA sequence or YP_001087135.1 for protein sequence). Both arehigh-molecular-mass proteins that bind to the surface of intestinalepithelial cells, where they are internalized and catalyze theglucosylation of cytoplasmic rho proteins, leading to cell death,inflammation and diarrhea. They have also been implicated in promotingC. difficile virulence, colonization, and neutrophil chemotaxis andactivation. The bacteria itself is not invasive and does not causetissue damage. By neutralizing the C. difficile toxins with antibodies,the pathogenic mechanism of the pathogen is blocked, its ability tothrive in the gut may be diminished, and the impact on the microbialecology could be minimized, allowing recovery of the normal microflora.The medical advantage of this approach could include more rapidrecovery, fewer relapses, and relief from selective pressure forantibiotic resistance in normal gut flora.

Accordingly, in an embodiment, the invention relates to a gram-positivebacterium as described herein, in which the polycistronic expressionunit comprises an antibody or fragment thereof, preferably a Fab, asdescribed herein elsewhere, directed against toxin A and/or toxin B ofClostridium. Most preferably, said antibody or fragment thereof is aneutralizing antibody. In a further embodiment, the invention relates toa gram-positive bacterium, preferably a Lactococcus sp. such asLactococcus lactis or an Enterococcus sp. such as Enterococcus faecalisor Enterococcus faecium, comprising a polycistronic expression unit,preferably integrated in the bacterial chromosome, said polycistronicexpression unit comprising an endogenous gene, preferably selected fromthe group consisting of eno, usp45, gap, pyk, rpmB and rplS, preferablyoriginating from a Lactococcus sp. or an Enterococcus sp., and one ormore exogenous genes encoding a neutralizing antibody or antibodyfragment, preferably a Fab, against toxin A and/or toxin B ofClostridium, preferably Clostridium dificile, said polycistronicexpression unit being preferably chromosomally integrated at the nativelocus of said endogenous gene, and said toxin A and/or toxin B antibody(fragment) gene preferably being transcriptionally coupled to the 3′ endof said endogenous gene, said transcriptional coupling preferably beingeffected by an intergenic region, preferably selected from the groupconsisting of intergenic regions preceding rplW, rplP, rpmD, rplB, rpsG,rpsE, rplN, rplM, rplE, and rplF of a gram-positive bacterium,preferably a Lactococcus sp. or a Enterococcus sp. The Clostridium toxinA and toxin B antibodies as described herein are known in the art (seee.g. Leung et al., J Pediatr 1991; 118(4 Pt 1):633-637; Wilcox. JAntimicrob Chemother 2004; 53(5):882-884; Sougioultzis et al.,Gastroenterology 2005; 128(3):764-770; Kyne et al., N Engl J Med 2000;342(6):390-397; Lowy et al., N Engl J Med; 362(3):197-205). Bothantibodies or fragments thereof may be located on separate polycistronicexpression units in the same or different gram-positive bacterium, butpreferably are located on a single polycistronic expression unit. Theinvention further relates to a method for preventing and/or treatingCDAD, comprising administering such gram-positive bacterium.

The skilled reader shall appreciate that the herein specifically reciteddiseases are only exemplary and their recitation is in no way intendedto confine the use of the reagents provided by the invention, e.g., thepromoters, nucleic acids, vectors and host cells of the invention, tothese particular diseases. Instead, a skilled reader understands thatthe reagents of the invention can be used to express in principle anyexpression products, preferably polypeptides, of interest, which may beof therapeutic relevance in not only the recited ones but also invarious further diseases or conditions of humans and animals.Consequently, once a suitable expression product, preferably apolypeptide, e.g., an antigen, antibody (fragment) and/or anon-vaccinogenic therapeutically active polypeptide, has been chosen ordetermined for a given ailment, a skilled person would be able toachieve its expression, isolation and/or delivery using the reagents ofthe invention.

The invention also contemplates treatment of diseases in other animalsincluding dogs, horses, cats and birds. Diseases in dogs include but arenot limited to canine distemper (paramyxovirus), canine hepatitis(adenovirus Cav-1), kennel cough or laryngotracheitis (adenovirusCav-2), infectious canine enteritis (coronavirus) and haemorrhagicenteritis (parvovirus).

Diseases in cats include but are not limited to viral rhinotracheitis(herpesvirus), feline caliciviral disease (calicivirus), felineinfectious peritonitis (parvovirus) and feline leukaemia (felineleukaemia virus). Other viral diseases in horses and birds are alsocontemplated as being treatable using the methods and compositions ofthe invention. To this purpose, the use of microorganisms expressingrecombinant interferons will be particularly preferred.

As used herein, the pharmaceutical composition preferably comprises atherapeutically effective amount of the gram-positive bacteria of theinvention and a pharmaceutically acceptable carrier, i.e., one or morepharmaceutically acceptable carrier substances and/or additives, e.g.,buffers, carriers, excipients, stabilisers, etc.

The term “pharmaceutically acceptable” as used herein is consistent withthe art and means compatible with the other ingredients of apharmaceutical composition and not deleterious to the recipient thereof.

The gram-positive bacteria of the invention can be suspended in apharmaceutical formulation for administration to the human or animalhaving the disease to be treated. Such pharmaceutical formulationsinclude but are not limited to live gram-positive bacteria and a mediumsuitable for administration. The gram-positive bacteria may belyophilized in the presence of common excipients such as lactose, othersugars, alkaline and/or alkali earth stearate, carbonate and/or sulphate(for example, magnesium stearate, sodium carbonate and sodium sulphate),kaolin, silica, flavorants and aromas. Gram-positive bacteriaso-lyophilized may be prepared in the form of capsules, tablets,granulates and powders (e.g. a mouth rinse powder), each of which may beadministered by the oral route. Alternatively, some gram-positivebacteria may be prepared as aqueous suspensions in suitable media, orlyophilized bacteria may be suspended in a suitable medium just prior touse, such medium including the excipients referred to herein and otherexcipients such as glucose, glycine and sodium saccharinate.

For oral administration, gastroresistant oral dosage forms may beformulated, which dosage forms may also include compounds providingcontrolled release of the gram-positive bacteria and thereby providecontrolled release of the desired protein encoded therein. For example,the oral dosage form (including capsules, tablets, pellets, granulates,powders) may be coated with a thin layer of excipient (usually polymers,cellulosic derivatives and/or lipophilic materials) that resistsdissolution or disruption in the stomach, but not in the intestine,thereby allowing transit through the stomach in favour ofdisintegration, dissolution and absorption in the intestine.

The oral dosage form may be designed to allow slow release of thegram-positive bacteria and of the produced exogenous proteins, forinstance as controlled release, sustained release, prolonged release,sustained action tablets or capsules. These dosage forms usually containconventional and well known excipients, such as lipophilic, polymeric,cellulosic, insoluble, swellable excipients. Controlled releaseformulations may also be used for any other delivery sites includingintestinal, colon, bioadhesion or sublingual delivery (i.e., dentalmucosal delivery) and bronchial delivery. When the compositions of theinvention are to be administered rectally or vaginally, pharmaceuticalformulations may include ointments, suppositories and creams. In thisinstance, the gram-positive bacteria are suspended in a mixture ofcommon excipients also including lipids. Each of the aforementionedformulations are well known in the art and are described, for example,in the following references: Hansel et al., Pharmaceutical dosage formsand drug delivery systems, 5th edition, William and Wilkins, 1990; Chien1992, Novel drug delivery system, 2nd edition, M. Dekker; Prescott etal. (1989, Novel drug delivery, J. Wiley & Sons); Cazzaniga et al,(1994, Oral delayed release system for colonic specific delivery, Int.J. Pharm.i08:7′.

Preferably, an enema formulation may be used for rectal administration.The term “enema” is used to cover liquid preparations intended forrectal use. The enema may be usually supplied in single-dose containersand contains one or more active substances dissolved or dispersed inwater, glycerol or macrogols or other suitable solvents.

Thus, according the invention, in a preferred embodiment, thegram-positive bacteria according to the invention as described hereinencoding a desired exogenous gene may be administered to the animal orhuman via mucosal, e.g., an oral, nasal, rectal, vaginal or bronchialroute by any one of the state-of-the art formulations applicable to thespecific route. Dosages of gram-positive bacteria for administrationwill vary depending upon any number of factors including the type ofbacteria and the gene encoded thereby, the type and severity of thedisease to be treated and the route of administration to be used.

Thus, precise dosages cannot be defined for each and every embodiment ofthe invention, but will be readily apparent to those skilled in the artonce armed with the present invention. The dosage could be anyhowdetermined on a case by case way by measuring the serum levelconcentrations of the recombinant protein after administration ofpredetermined numbers of cells, using well known methods, such as thoseknown as ELISA or Biacore (see examples). The analysis of the kineticprofile and half life of the delivered recombinant protein providessufficient information to allow the determination of an effective dosagerange for the transformed host cells.

In an embodiment, when the gram-positive bacteria according to theinvention as described herein express an antigen, the invention may thusalso provide a vaccine.

The term “vaccine” identifies a pharmaceutically acceptable compositionthat, when administered in an effective amount to an animal or humansubject, is capable of inducing antibodies to an immunogen comprised inthe vaccine and/or elicits protective immunity in the subject.

The vaccine of the invention would comprise the gram-positive bacteriaaccording to the invention as described herein and further optionally anexcipient. Such vaccines may also comprise an adjuvant, i.e., a compoundor composition that enhances the immune response to an antigen.Adjuvants include, but are not limited to, complete Freund's adjuvant,incomplete Freund's adjuvant, saponin, mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil or hydrocarbon emulsions, andpotentially useful pharmaceutically acceptable human adjuvants such asBCG (bacille Calmetle-Guerin) and Corynebacterium parvum.

It is thus made apparent that there have been provided in accordancewith the invention, biomarkers, uses and methods that provide forsubstantial advantages in the diagnosis, prediction, prognosis and/ormonitoring of impaired fracture healing. While the invention has beendescribed in conjunction with specific embodiments thereof, it isevident that many alternatives, modifications, and variations will beapparent to those skilled in the art in light of the foregoingdescription. Accordingly, it is intended to embrace all suchalternatives, modifications, and variations as follows in the spirit andbroad scope of the appended claims.

The aspects and embodiments of the invention are further supported bythe following non-limiting examples.

EXAMPLES Example 1 Selection of Intergenic Regions from the Lactococcuslactis Genome

Cellular proteins of an end-log culture of Lactococcus lactis ssp.cremoris strain MG1363 were visualized onto a protein gel with Coomassieblue staining, as indicated in FIG. 1. Lanes A and B contained 29 μg and58 μg MG1363 proteins, respectively. 12 defined proteins bands from laneA were isolated from the gel. Proteins were isolated and intergenicregions were identified by:

-   -   1) Identification of abundantly expressed proteins in the        fragments by partial peptide sequencing (MALDI-TOF/TOF) and        database search using combined peptide masses and sequence        information    -   2) Identification, using the chromosome sequence of Lactococcus        lactis ssp. cremoris strain MG1363 (Wegmann et al), of genes        encoding the abundantly expressed proteins (1) that are present        in an operon, but not as a “first gene”    -   3) Identification of intergenic regions that precede these        abundantly expressed genes

Table 3 lists thus identified intergenic regions. Underlined sequencesrepresent ribosome binding sites.

TABLE 3  2^(nd)  Function  Intergenic region gene 2^(nd) gene TAATG rplW50 S ribosomal  (SEQ ID NO: 1) protein L23 TAATCCATG rplP50 S ribosomal  (SEQ ID NO: 2) protein L16 TAAGGAGGAAAAAATG rpmD50 S ribosomal  (SEQ ID NO: 3) protein L30 TAATAGAGGAGGAAAATCGTG rplB50 S ribosomal  (SEQ ID NO: 4) protein L2 TAAGAAGGGAGATAAGTAAGAATG rpsG30 S ribosomal  (SEQ ID NO: 5) protein S7 TAAGGAAAGGGGTAATTAAACATG rpsE30 S ribosomal  (SEQ ID NO: 6) protein S5 TAAGCAAAACTAGGAGGAATATAGCATGrplN 50 S ribosomal  (SEQ ID NO: 7) protein L14

Example 2 Selection of Sites for Bicistronic Expression

Table 4 lists target promoters identified in Example 1 as driving highlevel expression. These promoters can be used as target sites forpolycistronic expression of exogenous genes.

TABLE 4 Band Gene as annotated in MG1363 Name 1 DNA-directed RNApolymerase, beta′ rpoC subunit/160 kD subunit DNA-directed RNApolymerase, beta rpoB subunit/140 kD subunit non-heme iron-bindingferritin dpsA 2 pyruvate kinase pyk glutamyl -tRNA synthetases gltX 3phosphopyruvate hydratase eno glutamine synthetase glnA glutaminesynthetase repressor glnR dipeptidase PepV pepV F0F1-type ATP synthasebeta subunit atpD (ATP synthase F1 beta subunit) F0F1-type ATP synthasealpha subunit atpA 4 multiple sugar-binding transport ATP- msmK bindingprotein acetoin dehydrogenase complex E1 pdhA component alpha subunit(acoA) cell division protein ftsA UDP-galactopyranose mutase glf13-phosphoglycerate kinase pgk glyceraldehyde-3-phosphate gapBdehydrogenase acetate kinase ackA1 3-oxoacyl-(acyl-carrier-protein)synthase fabF II 5 3-ketoacyl-(acyl-carrier-protein) fabG reductaseDNA-directed RNA polymerase, alpha rpoA subunit/40 kD subunit Prolinedipeptidase pepQ glutamyl aminopeptidase pepA predicted dehydrogenaserelated llmg_0272 protein 6 30S ribosomal protein S2 rpsB 50S ribosomalprotein L4 (rplD) rplD 50S ribosomal protein L23 rplW 50S ribosomalprotein L2 rplB Phenylalanyl-tRNA synthetase beta pheT chainfructose-bisphosphate aldolase fbaA 7 30S ribosomal protein S4 rpsDtranslation initiation factor 3 (IF-3) infC transcription elongationfactor GreA greA protease subunit of ATP-dependent Clp clpP proteasesuperoxide dismutase sodA 8 30S ribosomal protein S12 rpsL 30S ribosomalprotein S7 rpsG 50S ribosomal protein L18 rplR 30S ribosomal protein S5rpsE 50S ribosomal protein L30/L7E rpmD S-ribosylhomocysteinase luxS 50Sribosomal protein L15 rplO 50S ribosomal protein L11 rplK 9 30Sribosomal protein S8 rpsH 50S ribosomal protein L21 rplU 30S ribosomalprotein S13 rpsM 30S ribosomal protein S19 (rpsS) rpsS ribosomal proteinL22 (rplV) rplV ribosomal protein L16 (rplP) rplP ribosomal protein L14(rplN) rplN 30S ribosomal protein L19 rplS 30S ribosomal protein S11rpsK 10 30S ribosomal protein S10 rpsJ co-chaperonin GroES groES 50Sribosomal protein L24 rplX 50S ribosomal protein L10 rplJ 50S ribosomalprotein L7/L12 rplL 11 HU-like DNA-binding protein hllA 50S ribosomalprotein L28 rpmB phosphotransferase system IIB ptcB component

The β-glucuronidase (uidA) gene from E. coli was introduced as reportergene in Lactococcus lactis MG1363. The uidA gene productβ-glucuronidase, catalyses the cleavage of a wide variety ofβ-glucuronides that are commercially available as histochemical andspectrophotometric substrates. Strain sAGX0090 has the PhIIA>>uidAexpression cassette at the thyA locus (FIG. 2). This promoter was alsoused in strains sAGX0037 and sAGX0085.

As depicted in FIG. 2, dual cistron constructs were made by insertinguidA in the Lactococcus lactis MG1363 chromosome at the 3′ end ofseveral endogenous genes (gene X). Hereby, rpmD was used as intergenicsequence between the endogenous genes of interest from Table 4 and uidAto identify sites that result in higher β-glucuronidase (GUS) activitycompared to sAGX0090.

Lactococcus lactis cultures were grown for 16 hours at 30° C. in GM17supplemented with thymidine when needed. Cells of 1 ml culture werewashed and resuspended in 1 ml demineralized water. Cells were disruptedwith MP Biomedicals lysing matrix B and Fasprep-24 device at 6 m/s for40 seconds. Tubes were centrifuged and a dilution series of the cellsupernatant was made. GUS activity was measured by adding p-nitrophenylsubstrate and β-mercaptoethanol which gives the solution a yellow colourupon presence of β-glucuronidase. GUS activity was measured at 405 nmand expressed relatively to reference strain sAGX0090. All strains weretreated in parallel.

FIG. 3 shows relative GUS-activity of gene X>>rpmD>>uidA dual cistronconstructs. GUS-activity is expressed relatively to reference strainsAGX0090 that carries the PhIIA>>uidA expression cassette and isindicated on the Y-axis. GUS-activity in all dual cistron strains wasfound to be higher than the reference strain. In particular,GUS-activity in sAGX0168 (enoA>>rpmD>>uidA) and sAGX0222(gapB>>rpmD>>uidA) was found to be 6.89 and 20.99 times higher whencompared to sAGX0090, respectively.

These results clearly confirm that bicistronic expression allowsenhanced protein expression levels over a wide variety of settings.

Example 3 Bicistronic Expression of Human Pro-insulin

The usp45 secretion leader (SS) was fused to human pro-insulin (ins) toobtain secretion of pro-insulin (SS::ins). The [SS::ins] expressioncassette was either integrated in the Lactococcus lactis MG1363chromosome at the thyA locus and expressed directly from PthyA(sAGX0121) or was inserted, along with the rpmD intergenic regionpreceding SS::ins, as a second cistron downstream from usp45 (sAGX0121)or enoA (sAGX0164) (FIG. 4a ). The insulin secretion capacity wasquantified by ELISA to evaluate bicistronic expression of cargo comparedto PthyA driven expression at the thyA locus.

Attempts to construct a PhIIA>>SS::Ins integration plasmid have failed.

Strains were inoculated from single colony into 2 ml GM17 supplementedwith 200 μM thymidine when needed and grown for 16 hours at 30° C. Forthe quantification of pro-insulin secretion, these saturated overnightcultures were diluted 1/25 in 5 ml fresh GM17 medium and grown for 4hours at 30° C. Cells were collected by centrifugation at 3220×g for 10minutes, resuspended in an equal amount BAM9 medium and cultured foranother 3 hours at 30° C. BAM9 contains M9 salts, 0.5% aminoplasmal,0.5% glucose, 25 mM NaHCO₃, 25 mM Na₂CO₃, 2 mM MgSO₄, 0.1 mM CaCl₂ and200 μM thymidine. Cells and culture supernatants were separated bycentrifugation at 3220×g for 10 minutes. The amount of secreted humanpro-insulin in the culture supernatant was quantified by ELISA providedby Mercodia. All strains were treated in parallel.

FIG. 4b represents the quantification of human pro-insulin secretion byLactococcus lactis strains sAGX0122, sAGX0121 and .sAGX0164, The amountof secreted pro-insulin was expressed as ng/ml and indicated on theY-axis. The figure clearly demonstrates that strains comprising abicistronic expression cassette have a significantly higher cargoexpression than reference strain. In particular, insulin secretion washighest when SS::ins was coupled through rpmD to enoA.

Example 4 Bicistronic Expression of cA2 Fab

Dual cistron expression constructs were generated with heavy chain andlight chain of cA2 anti-hTNF Fab. All expression units are driven by thethyA promoter and are located on plasmids. All carry genes for the lightchain, VLCL (L) and the Fab fragment of the heavy chain, VHCH1 (H),derived from the cA2 infliximab monoclonal antibody. L>>H and H>>Lconfigurations are coupled by intergenic regions preceding rpmD, rplB,rpsG, rpsE or rplN. All constructs are plasmid borne.

FIG. 5 reveals that both heavy chain and light chains were highlyexpressed by the dual cistron constructs, leading to high levels offunctional cA2 anti-TNF Fab. FIG. 5 further reveals that cA2 anti-TNFexpression increased when the heavy chain was positioned before thelight chain, irrespective of the intergenic region.

For the quantification of anti-hTNF secretion, strains were inoculatedfrom single colony into 2 ml GM17 and grown for 16 hours at 30° C. Thesesaturated overnight cultures were diluted 1/25 in 5 ml fresh GM17 mediumand grown for 4 hours at 30° C. Cells were collected by centrifugationat 3220×g for 10 minutes, resuspended in an equal amount BAM9 medium andcultured for another 3 hours at 30° C. BAM9 contains M9 salts, 0.5%aminoplasmal, 0.5% glucose, 25 mM NaHCO₃, 25 mM Na₂CO₃, 2 mM MgSO₄, 0.1mM CaCl₂ and 200 μM thymidine. Cells and culture supernatants wereseparated by centrifugation at 3220×g for 10 minutes. Crude supernatantsfrom strains carrying the individual constructs were prepared inparallel and were assayed for the presence of anti-TNF activity. Thiswas done by direct ELISA using human TNF as capture protein. VLCLportions were detected by rabbit anti-human IgG antiserum and revealedby alkaline phosphatase conjugated anti-rabbit antiserum. Phosphataseactivity was measured by colorimetric assay and read out as OD402. Allstrains were treated in parallel.

Example 5 Bicistronic Expression of CDP870

Dual cistron expression constructs were generated with heavy chain andlight chain of CDP870 anti-TNF Fab. All expression units are located onthe bacterial chromosome.

FIG. 6a : CDP870 light and heavy chain Fab fusions to usp45 secretionleader encoding sequences (SS::CDP870 VLCL and SS::CDP870 VHCH1) wereinserted as a second and third cistron downstream from usp45 (sAGX0219,sAGX0220). In sAGX0219 and sAGX0220, rpmD was used to couple SS::CD870genes to usp45. To avoid genetic instability, light and heavy chaingenes were coupled through the intergenic region preceding rplN. InsAGX0219, the light chain gene precedes the heavy chain gene, while insAGX0220, the heavy chain gene precedes the light chain gene.

For the quantification of anti-hTNF secretion, strains were inoculatedfrom single colony into 2 ml GM17 and grown for 16 hours at 30° C. Thesesaturated overnight cultures were diluted 1/25 in 5 ml fresh GM17 mediumand grown for 4 hours at 30° C. Cells were collected by centrifugationat 3220×g for 10 minutes, resuspended in an equal amount BAM9 medium andcultured for another 3 hours at 30° C. BAM9 contains M9 salts, 0.5%aminoplasmal, 0.5% glucose, 25 mM NaHCO₃, 25 mM Na₂CO₃, 2 mM MgSO₄, 0.1mM CaCl₂ and 200 μM thymidine. Cells and culture supernatants wereseparated by centrifugation at 3220×g for 10 minutes. Crude supernatantsfrom strains carrying the individual constructs were prepared inparallel and were assayed for the presence of anti-TNF activity. Thiswas done by direct ELISA using human TNF as capture protein withRemicade as a reference standard. VLCL portions were detected by rabbitanti-human IgG antiserum and revealed by alkaline phosphatase conjugatedanti-rabbit antiserum. Phosphatase activity was measured by colorimetricassay and read out as OD402. All strains were treated in parallel.

FIG. 6b reveals that both heavy chain and light chains were highlyexpressed by the dual cistron constructs, leading to high levels offunctional CDP870 anti-TNF Fab. FIG. 6b further reveals that CDP870anti-TNF expression substantially increased when the heavy chain waspositioned before the light chain.

Example 6 Bicistronic Expression of Human Trefoil Factor 1 (hTFF1)

Expression constructs were generated with the usp45 secretion leadercoding sequence fused to hTFF1 (SS::hTFF1). All expression units arelocated on the bacterial chromosome. It was not possible to constructintegration plasmids for monocistronic hTFF1 expression using strongerpromoters than PhIIA.

FIG. 7a : The usp45 secretion leader coding sequence (SS) was fused tohTFF1 to obtain secretion of hTFF1 (SS::hTFF1). The SS::hTFF1 expressioncassette was either integrated in the Lactococcus lactis MG1363chromosome at the thyA locus and expressed directly from PhIIA(sAGX0085) or was inserted, along with the rpmD intergenic regionpreceding SS::hTFF1, as a second cistron downstream from gapB(sAGX0276).

FIG. 7b : The hTFF1 secretion capacity was quantified by ELISA toevaluate bicistronic expression of cargo compared to PhIIA drivenexpression at the thyA locus.

For the quantification of hTFF1 secretion, strains were inoculated fromsingle colony into 2 ml GM17 and grown for 16 hours at 30° C. Thesesaturated overnight cultures were diluted 1/25 in 5 ml fresh GM17 mediumand grown for 4 hours at 30° C. Cells were collected by centrifugationat 3220×g for 10 minutes, resuspended in an equal amount BAM9 medium andcultured for another 3 hours at 30° C. BAM9 contains M9 salts, 0.5%aminoplasmal, 0.5% glucose, 25 mM NaHCO₃, 25 mM Na₂CO₃, 2 mM MgSO₄, 0.1mM CaCl₂ and 200 μM thymidine. At this stage, colony forming units (CFU)of all cultures were determined. Cells and culture supernatants wereseparated by centrifugation at 3220×g for 10 minutes. Crude supernatantsfrom strains carrying the individual constructs were prepared inparallel and were assayed by ELISA using purified hTFF1 as a referencestandard. The amount of secreted hTFF1 was expressed as ng/ml and ng/10⁹CFU. All strains were treated in parallel.

FIG. 7b clearly demonstrates that the strain comprising a bicistronicexpression cassette (sAGX0276) has a significantly higher cargoexpression than reference strain (sAGX0085). The amount of secretedhTFF1 expression was substantially enhanced (>5 fold per ml; >12 foldper CFU) when hTFF1 was coupled through rpmD to gapB.

Example 7 Selection of Intergenic Regions from the Enterococcus faeciumGenome

Cellular proteins of an end-log culture of Enterococcus faecium strainLMG 15709 (Enterococcus faecium [Orla-Jensen 1919] Schleifer andKilpper-Bälz 1984 VP; LMG 15709; ATCC 6057; DSM 2146; NCIMB 8842) werevisualized onto a protein gel with Coomassie blue staining, as indicatedin FIG. 8. Lanes A, B and C contained the cellular proteins of the lysedcell equivalent of 284 μl, 142 μl and 56.8 μl end-log culture ofEntercococcus faecium LMG15709, respectively. 12 defined proteins bandsfrom lane C were isolated from the gel. Proteins were isolated andintergenic regions were identified by:

-   -   1) Identification of abundantly expressed proteins in the        fragments by partial peptide sequencing (MALDI-TOF/TOF) and        database search using combined peptide masses and sequence        information.    -   2) Identification, using the chromosome sequence of Enterococcus        faecium PC4.1 (retrieved form the NCBI Genome databank, GenBank        accession number ADMM01000000), of genes encoding the abundantly        expressed proteins (1) that are present in an operon, but not as        a “first gene”.    -   3) Identification of intergenic regions that precede these        abundantly expressed genes (Table 5).

Table 5 lists identified intergenic regions in Enterococcus faeciumLMG15709. Underlined sequences represent ribosome binding sites.

TABLE 5  2^(nd)  Function  Intergenic region gene 2^(nd) gene TAATC rplP50 S ribosomal  (SEQ ID NO: 8) protein L16 TAAGGAGGACAACAATA rpmD50 S ribosomal  (SEQ ID NO: 9) protein L30 TAATAGGAGGGAATTTCA rplM50 S ribosomal  (SEQ ID NO: 10) protein L13 TTAGAAGAAGGAGGAATACCATTCrpsE 30 S ribosomal  (SEQ ID NO: 11) protein S5TAAAAGTTTAAGGAAGGAGGGTCTTACTGA rplE 50 S ribosomal  (SEQ ID NO: 12)protein L5 TAATCAAGTAGAATCTACAAGGAGGTGTCT rplF 50 S ribosomal TTAA (SEQ ID NO: 13) protein L6

Example 8 Selection of Sites in the Enterococcus faecium Genome forBicistronic Expression

Table 6 lists highly expressed Enterococcus faecium genes identified inExample 7 as driving high level expression. Promoters driving thesegenes can be used as target sites for polycistronic expression ofexogenous genes. These endogenous genes can further be used as firstgene in a polycistronic expression module, transcriptionally ortranslationally coupled, through an intergenic region, to downstreamexogenous genes.

TABLE 6 Band Gene annotation Name 1 Enolase [Enterococcus faecium DO];gi|69249235 eno Elongation factor Tu [Enterococcus faecium TX13330];gi|227550718 tuf 2 Glyceraldehyde-3-phosphate dehydrogenase[Enterococcus faecium gap TX1330]; gi|227552066 3 L-lactatedehydrogenase [Enterococcus faecium DO]; gi|69245441 ldh Aspartatecarbamoyltransferase [Enterococcus faecium DO]; pyrB gi|69247601Ribose-phosphate pyrophosphokinase [Enterococcus faecium DO];gi|69245416 4 Pyruvate kinase [Enterococcus faecium DO]; gi|69247355 pykOligoendopeptidase F [Enterococcus faecium E1039]; gi|293553061 pepFAspartyl-tRNA synthetase bacterial/mitochondrial type [Enterococcus aspSfaecium DO]; gi|69247937 5 Lysyl-tRNA synthetase [Enterococcus faeciumTX1330]; gi|227552660 lysS GroEL [Enterococcus faecium]; gi|35187728groEL 6 Phosphoglycerate kinase [Enterococcus faecium E1039];gi|293557157 pgk 7 Fructose-bisphosphate aldolase class-II [Enterococcusfaecium 1,230,933]; gi|293557157 2,3-bisphosphoglycerate-dependentphosphoglycerate mutase [Enterococcus faecium E1039]; gi|293556592Saicar synthetase [Enterococcus faecium 1,141,733]; gi|257887626 purC 82,3-bisphosphoglycerate-dependent phosphoglycerate mutase [Enterococcusfaecium E1039]; gi|293556592 Saicar synthetase [Enterococcus faecium1,231,501]; gi|257884790 purC 9 50S ribosomal protein L5 [Enterococcusfaecium DO]; gi|69247181 rplE 50S ribosomal protein L6 [Enterococcusfaecium DO]; gi|69247184 rplF Peroxiredoxin [Enterococcus faeciumTX1330]; gi|227551517 aphC Xanthine phosphoribosyltransferase[Enterococcus faecium 1,230,933]; gi|257878081 10 Elongation factor G[Enterococcus faecium TX1330]; gi|227550717 fusA 11 30S ribosomalprotein S5, bacterial and organelle form [Enterococcus rpsE faecium DO];gi|69247186 50S ribosomal protein L16 [Enterococcus faecium]; gi|9931590rplP Universal stress protein family [Enterococcus faecium E980];gi|293571359 Ferritin [Enterococcus faecium 1,230,933]]; gi|25788041330S ribosomal protein S7 [Enterococcus faecium TX1330]; rpsGgi|227550716 50S ribosomal protein L13 [Enterococcus faecium 1,230,933];rplM gi|257880414 12 M20 family peptidase PepV [Enterococcus faeciumTX1330]; pepV gi|227550917 Glutamyl-tRNA synthetasebacterial/mitochondrial [Enterococcus faecium gltX DO]; gi|69245495 Celldivision protein FtsA [Enterococcus faecium DO]; gi|69244711 ftsAAsparaginyl-tRNA synthetase, class IIb [Enterococcus faecium DO]; asnCgi|69247321

In such way, the β-glucuronidase (uidA) gene from E. coli was introducedas reporter gene in Enterococcus faecium LMG15709. The uidA gene productβ-glucuronidase, catalyses the cleavage of a wide variety ofβ-glucuronides that are commercially available as histochemical andspectrophotometric substrates. As depicted in FIG. 9, dual cistronconstructs were made by inserting uidA in the Enterococcus faeciumLMG15709 chromosome at the 3′ end of several endogenous genes (gene X,gap and eno in this example; FIG. 9). Hereby, rpmD of Enterococcusfaecium was used as intergenic region between the endogenous genes ofinterest from Table 6 and uidA to identify sites that result in highestβ-glucuronidase (GUS) activity (FIG. 10).

Enterococcus faecium cultures were grown for 16 hours at 30° C. in GM17supplemented with thymidine. Cells of 1 ml culture were washed andresuspended in 1 ml demineralized water. Cells were disrupted with MPBiomedicals lysing matrix B and Fasprep-24 device at 6 m/s for 40seconds. Tubes were centrifuged and a dilution series of the cellsupernatant was made. GUS activity was measured by adding p-nitrophenylsubstrate and β-mercaptoethanol which gives the solution a yellow colourupon presence of β-glucuronidase. GUS activity was measured at 405 nmand expressed relatively to reference Lactococcus lactis strainsAGX0090. All strains were treated in parallel.

FIG. 10 shows relative GUS-activity of gene X>>rpmD>>uidA dual cistronconstructs. GUS-activity is expressed relatively to reference strainsAGX0090 that carries the PhIIA>>uidA expression cassette and isindicated on the Y-axis. GUS-activity in all dual cistron strains wasfound to be higher than the reference strain.

In particular, GUS-activity in sAGX0270 (gap>>rpmD>>uidA) and sAGX0271(eno>>rpmD>>uidA) was found to be 30.6 and 26.9 times higher whencompared to sAGX0090, respectively.

These results clearly confirm that bicistronic expression allowsenhanced protein expression levels over a wide variety of settings.

Example 9 Bicistronic Expression of Human Interleukin-10 (hIL10) byEnterococcus faecium

The DNA coding sequence of the usp45 secretion leader of Lactococcuslactis (SS) was fused in frame to the DNA sequence of mature hIL10 toobtain secretion of hIL10. The [SS::hIL10] expression cassette wasinserted, along with the rpmD intergenic region of Enterococcus faeciumpreceding SS::hIL10, as a second cistron downstream from gap (sAGX0279;FIG. 11a ). The hIL10 secretion capacity was quantified by ELISA toevaluate bicistronic expression of cargo in Enterococcus faecium.Enterococcus faecium sAGX0270 served as a negative control.

Strains were inoculated from single colony into 10 ml GM17 supplementedwith 200 μM thymidine (GM17T) when needed and grown for 16 hours at 30°C. For the quantification of hIL10 secretion, these saturated overnightcultures were diluted 1/25 in 5 ml fresh GM17T medium and grown for 4hours at 30° C. Cells were collected by centrifugation at 3220×g for 10minutes, resuspended in an equal amount BAM9T medium and cultured foranother 3 hours at 30° C. BAM9T contains M9 salts, 0.5% aminoplasmal,0.5% glucose, 25 mM NaHCO₃, 25 mM Na₂CO₃, 2 mM MgSO₄, 0.1 mM CaCl₂ and200 μM thymidine. Cells and culture supernatants were separated bycentrifugation at 3220×g for 10 minutes. The amount of secreted humanhIL10 in the culture supernatant was quantified by sandwich hIL10 ELISA.All strains were treated in parallel.

FIG. 11b represents the quantification of human hIL10 secretion byEnterococcus faecium strains sAGX0270 and sAGX0279 The amount ofsecreted hIL10 was expressed as ng/10⁹ CFU cells in 3 hours andindicated on the Y-axis. The figure clearly demonstrates thatEnterococcus faecium strains comprising a bicistronic expressioncassette are able to secrete considerable amounts of the cargo proteinhIL10.

Example 10 Bicistronic Expression of Human Interleukin-27 (hIL27) byEnterococcus faecium

The DNA coding sequence of the usp45 secretion leader of Lactococcuslactis (SS) was fused in frame to the DNA sequence of mature hIL27 toobtain secretion of hIL27. The [SS::hIL27] expression cassette wasinserted, along with the rpmD intergenic region of Enterococcus faeciumpreceding SS::hIL27, as a second cistron downstream from gap (sAGX0317;FIG. 12a ). The hIL27 secretion capacity was quantified by ELISA toevaluate bicistronic expression of hIL27. Enterococcus faecium sAGX0270served as a negative control,

Strains were inoculated from single colony into 10 ml GM17 supplementedwith 200 μM thymidine (GM17T) and grown for 16 hours at 30° C. For thequantification of hIL27 secretion, these saturated overnight cultureswere diluted 1/25 in 5 ml fresh GM17T medium and grown for 4 hours at30° C. Cells were collected by centrifugation at 3220×g for 10 minutes,resuspended in an equal amount BM9T medium and cultured for another 3hours at 30° C. BM9T contains M9 salts, 0.5% casitone, 0.5% glucose, 25mM NaHCO₃, 25 mM Na₂CO₃, 2 mM MgSO₄, 0.1 mM CaCl₂ and 200 μM thymidine.Cells and culture supernatants were separated by centrifugation at3220×g for 10 minutes. The amount of secreted human hIL27 in the culturesupernatant was quantified by sandwich hIL27 ELISA (R&D systems). Allstrains were treated in parallel.

FIG. 12b represents the quantification of human hIL27 secretion byEnterococcus faecium strains sAGX0270 and sAGX0317. The amount ofsecreted hIL27 was expressed as ng/10⁹ CFU cells in 3 hours andindicated on the Y-axis. The figure clearly demonstrates thatEnterococcus faecium strain sAGX0317 comprising a bicistronic expressioncassette positioned downstream of the Enterococcus faecium gap gene isable to efficiently secrete considerable amounts of the exogenousprotein hIL27.

Example 11 Bicistronic Expression of CDP870 Fab by Enterococcus faecium

A dual cistron expression constructs was generated with heavy chain andlight chain genes of CDP870 Fab. All expression units are located on thebacterial chromosome.

FIG. 13a : CDP870 heavy and light chain Fab fusions to the Lactococcuslactis MG1363 usp45 secretion leader (SS) encoding sequences [SS::CDP870VHCH1] and [SS::CDP870 VLCL] were inserted as a second and third cistrondownstream from the Enterococcus faecium LMG15709 gap gene (sAGX0278).In sAGX0278, the intergenic region of Enterococcus faecium rpmD was usedto couple SS::CD870 expression cassettes to gap. To avoid geneticinstability, heavy and light chain genes were coupled through theintergenic region preceding Lactococcus lactis rpmD and different codonusage was used in the usp45 secretion signals of the two SS::CD870expression cassettes The heavy expression cassette precedes the lightchain expression cassette in sAGX0278.

For the quantification of CDP870 fab secretion, strains were inoculatedfrom single colony into 10 ml GM17T and grown for 16 hours at 30° C.These saturated overnight cultures were diluted 1/25 in 5 ml fresh GM17Tmedium and grown for 4 hours at 30° C. Cells were collected bycentrifugation at 3220×g for 10 minutes, resuspended in an equal amountBAM9T medium and cultured for another 3 hours at 30° C. BAM9T containsM9 salts, 0.5% aminoplasmal, 0.5% glucose, 25 mM NaHCO₃, 25 mM Na₂CO₃, 2mM MgSO₄, 0.1 mM CaCl₂ and 200 μM thymidine. Cells and culturesupernatants were separated by centrifugation at 3220×g for 10 minutes.Crude supernatants from strains carrying the individual constructs wereprepared in parallel and were assayed for the presence of TNF bindingactivity. This was done by direct ELISA using human TNF as captureprotein with Cimzia® (CDP870 fab linked to PEG) as a reference standard.CDP870 fab was detected by goat anti-human Fab antiserum and revealed byHRP conjugated donkey anti-goat IgG(H+L) antiserum. HRP activity wasvisualized by TMB substrate. Reaction was stopped after 30 minutes byadding HCl. Absorbance was measured at 450 nm with 595 nm as referencewavelength. All strains were treated in parallel.

FIG. 13b reveals that both heavy chain and light chains were expressedby the multiple cistron construct in Enterococcus faecium, leading tothe secretion of functional CDP870 anti-TNF Fab.

Example 12 Anti-hTNF Producing L. lactis Bacteria (According to theInvention) Protects Against hTNF-Induced Intestinal Damage in A20IEC-KOMice

Generation of Tissue-specific A20 Deficient Mice.

Conditional A20/tnfaip3 knockout mice, in which exons IV and V oftnfaip3 gene are flanked by two LoxP sites, were generated as described(Piguet et al., 1999, Lab Invest 79, 495-500). A20 floxed mice werecrossed with Villin-Cre transgenic mice generating IEC-specific A20knockout mice (A20^(IEC-KO)) (Madison et al., 2002, J Biol Chem 277,33275-33283). Experiments were performed on mice backcrossed into theC57BL/6 genetic background for at least four generations.

In Vivo TNF Toxicity.

Mice were injected i.p. with different doses hTNF (50, 10, 8, 6, 4 and 2μg hTNF/20 g body weight). E. coli-derived recombinant hTNF had aspecific activity of 6.8×10⁷ IU/mg. Human TNF was produced and purifiedto homogeneity in our laboratory, and endotoxin levels did not exceed 1ng/mg of protein. hTNF injection in A20IEC-KO mice induces severalpathological and immunological changes that are indicative of intestinaldamage. Low dose hTNF does not induce severe systemic effects andlethality, but induces pro-inflammatory cytokine and chemokineexpression which can be measured both in serum and homogenates ofintestinal tissue. Mice were euthanized after 4 or 5 h for histologicalanalysis. For therapeutic studies, A20^(IEC-KO) mice received anti-TNFproducing L. lactis bacteria (according to the invention) by oral gavage5 times (5×10¹⁰ CFU with 30 min interval) prior to an intraperitonealinjection with hTNF (2 μg and 6 μg hTNF/20 g body weight). Controlgroups were either treated with the parental L. lactis strain MG1363 orwith bacterial medium BM9T (vehicle). In addition, a positive controlgroup was treated with a single injection of Remicade (30 mg/kg). Bodytemperatures were monitored every hour.

Tissue Sample Preparation.

Freshly isolated colonic and ileal segments were flushed with PBS toremove the fecal content and subsequently flushed with formalin (4%formaldehyde in phosphate buffered saline (PBS)) and fixed by incubationovernight in a 10-fold excess of formalin at 4° C. The formalin wasremoved and intestines were washed twice with PBS prior to embedding inparaffin wax using standard methods.

Histology.

Tissue sections of 4 μm were cut and stained with hematoxylin/eosinusing standard techniques. For combined Alcian Blue (AB) and PASstainings, dewaxed sections were hydrated to distilled water andincubated in Alcian Bleu for 20 min. Sections were subsequentlyincubated in 1% periodic acid for 10 min followed by incubation inSchiff's reagent for 10 min. Nuclei were counterstained with Mayer'sHaematoxylin for 30 sec. Alkaline phosphatase detection was performed byincubation of dewaxed and hydrated tissue sections in NBT/BCIP solutionin the dark (70 μl NBT+70 μl BCIP+4860 μl Buffer A, NBT=1.5% NitroblueTetrazolium Chloride solution in 70% dimethyl formamide, BCIP=1%5-bromo-4-Cloro-3-Indolyl Phosphatase in 100% dimethyl formamide, BufferA=0.1M TrisHCl, 0.1M NaCl, 0.05M MgCl2, pH9.5). For immunochemistry,sections were dewaxed and incubated in Dako antigen retrieval solutionand boiled for 20 min in a Pick cell cooking unit and cooled down for2.5 h. Endogenous peroxidase activity was blocked by immersing slides inperoxidase-blocking buffer (0.040 M citric acid, 0.121 M disodiumhydrogen phosphate, 0.030 M sodium azide, 1.5% hydrogen peroxide) for 15min at room temperature. Blocking buffer (1% bovine serum albumin inPBS) was added to the slides for 30 min at room temperature. Primaryantibodies (rabbit anti-lysozyme; 1:1,750 dilution—Dako; rabbitanti-mucin-2; 1:500 22) were added in blocking buffer and tissuesections were incubated overnight. Secondary antibody was added (polymerhorseradish peroxidase-labelled anti-rabbit, Envision) for 30 min atroom temperature. Peroxidase was detected by adding diaminobuteric acid(DAB) substrate for 10 min at room temperature and nuclei werecounterstained with Mayer's haematoxylin for 2 min. Microscopicmeasurement of Paneth cell granule radii was done with Leica Imagemanager 500 software.

Quantification of Cytokines.

Cytokines and chemokines in serum and tissue homogenates were quantifiedby Cytometric Bead Array kits (CBA) (BD Biosciences) on a FACS Caliburcytometer equipped with CellQuest Pro and CBA software (BD Biosciences).

Quantitative Real-time PCR.

Ileal segments of 5 cm long were freshly isolated and flushed with PBSto remove the fecal content. One end was ligated and segments werefilled with RNA lysis buffer (Aurum Total RNA Mini kit, Bio-RadLaboratories) and incubated on ice for 5 min. RNA was purified from thelysate solution using the Aurum Total RNA Mini kit (Bio-RadLaboratories). cDNA synthesis was performed using the iScript cDNAsynthesis kit (Bio-Rad Laboratories) according to the manufacturer'sinstructions. 10 ng cDNA was used for quantitative PCR in a total volumeof 10 μl with LightCycler 480 SYBR Green I Master Mix (Roche) andspecific primers on a LightCycler 480 (Roche). Real-time PCR reactionswere performed in triplicates. The following mouse-specific primers wereused: ubiquitin forward, 5′-AGGTCAAACAGGAAGACAGACGTA-3′ (SEQ ID NO: 14);and ubiquitin reverse, 5′-TCACACCCAAGAACAAGCACA-3′ (SEQ ID NO: 15).Lysozyme-P forward, 5′-GCCAAGGTCTAACAATCGTTGTGAGTTG-3′ (SEQ ID NO: 16);Lysozyme-P reverse, 5′-CAGTCAGCCAGCTTGACACCACG-3′ (SEQ ID NO: 17);Cryptidin-1 forward, 5′-TCAAGAGGCTGCAAAGGAAGAGAAC-3′ (SEQ ID NO: 18);Cryptidin-1 reverse, 5′-TGGTCTCCATGTTCAGCGACAGC-3′ (SEQ ID NO: 19).

Statistical Analyses.

Results are expressed as the mean±SEM. Statistical significance betweenexperimental groups was assessed using an unpaired two-sample Studentt-Test.

Characterization of Anti-hTNF Producing L. lactis Bacteria to ProtectAgainst hTNF-induced Pathological and Immunological Alterations inA20IEC-KO Mice.

Both control groups injected with 2 μg hTNF showed a more drastic dropin body temperature compared to A20IEC-KO mice treated with anti-hTNFproducing L. lactis bacteria (FIG. 14a , left panel). The protectiveeffect of sAGX0220 treatment observed in mice treated with 2 μg hTNFcould no longer be observed when mice were injected with a higherconcentration of hTNF (6 μg), whereas Remicade treatment clearly had aprotective effect (FIG. 14a , right panel). sAGX0220 pretreatmentsignificantly reduced MCP-1, KC and IL-6 levels in ileal and colonhomogenates and in serum of mice treated with either 2 μg hTNF, comparedto vehicle-treated mice (FIG. 14b,c ). However, there was a comparablereduction in mice treated with the parental L. lactis strain MG1363,indicating that the administration of bacteria on itself can alreadyhave a probiotic protective effect. Mice challenged with 6 μg hTNFshowed a similar reduction in serum and tissue cytokine and chemokinelevels upon sAGX0220 pretreatment, to the same extent as Remicade does(FIG. 14d,e ). In this setting, however, the protective effect of theparental MG1363 strain is mostly lost (FIG. 14d,e ).

Example 13 Passive Immunization Against Clostridium difficile-associatedDisease with Toxin-Neutralizing Antibodies Locally Produced and SecretedVia Lactococcus lactis

a) Gene Synthesis of VLCL and VHCH1 of C. difficile Toxin A and C.difficile Toxin B Neutralizing mAb

De novo gene synthesis (optimized for L. lactis codon usage) isperformed based on the VH and VL amino acid sequence informationaccording to a routine method (Stemmer et al., Gene 1995; 164(1):49-53).This method makes use of synthetic 40-mer oligonucleotides that span theentire coding as well as the non-coding strand, in such way that everyoligonucleotide from the coding strand will be 100% complementary withrespectively the last and first 20 base pairs of two consecutiveoligonucleotides in the non-coding strand, and vice versa.

Genetic information that encodes the secretion leader of L. lactisunidentified secreted 45-kDa protein (Usp45) is added to the 5′ end ofVLCL and VHCH1 genes.

To guarantee coordinated expression, these synthetic VLCL and VHCH1genes are placed in tandem and joined by the rpmD intergenic region.This leads to the formation of functional VLCL>>VHCH1 operons. Alsoconstructed are variants of the above described synthetic genes thatalso encode C-terminal E and FLAG peptide tags. This visualizes fullsize and/or potential degradation products, and enables the verificationof light chain and heavy chain assembly and toxin binding.

The resulting gene constructs will be sequence verified.

b) Construction of L. lactis Strains Secreting Toxin A/B NeutralizingFab: Integration at the Usp45 Locus

This task consists of the following stages:

-   -   1. Construction of integration vectors for the integration        downstream the usp45 locus of        -   VLCL>>VHCH1 of C. difficile toxin A neutralizing Mab        -   VLCL>>VHCH1 of C. difficile toxin B neutralizing Mab        -   3′ E and FLAG tagged variants of the above

The synthetic VLCL>>VHCH1 operons are flanked at the 5′ end by 1 kb ofthe 3′ end of the L. lactis usp45 gene followed by the rplN intergenicregion. The VLCL>>VHCH1 operons are flanked at the 3′ end by a 1 kbfragment of the 3′ downstream flanking region of the L. lactis usp45gene. Constructions are made by overlap PCR DNA annealing. The resultingplasmids are sequence verified.

-   -   2. The above generated integration plasmids are used for        integration via translational coupling to the usp45 gene of L.        lactis MG1363. Integration is performed by double homologous        recombination at both 5′ and 3′ flanking regions (corresponding        to 1 kb regions flanking the 3′ end of usp45), and verified by        PCR and DNA sequencing.        c) Construction of L. lactis Strains Secreting Toxin A/B        Neutralizing Fab: Integration at the enoA Locus

This task consists of the following stages:

-   -   1. Construction of integration vectors for the integration        downstream of the enoA locus of        -   VLCL>>VHCH1 of C. difficile toxin A neutralizing Mab        -   VLCL>>VHCH1 of C. difficile toxin B neutralizing Mab        -   3′ E and FLAG tagged variants of the above

The synthetic VLCL>>VHCH1 operons from Task 7 are flanked at the 5′ endby 1 Kb of the 3′ end of the L. lactis enoA gene followed by the rplNintergenic region. The VLCL>>VHCH1 operons are flanked at the 3′ end bya 1 Kb fragment of the 3′ downstream flanking region of the L. lactisenoA gene. Constructions are made by overlap PCR DNA annealing. Theresulting plasmids are sequence verified.

-   -   2 The above generated integration plasmids are used for        integration via translational coupling to the enoA gene of L.        lactis MG1363. Integration is performed by double homologous        recombination at both the 5′ and 3′ flanking regions        (corresponding to 1 kb regions flanking the 3′ end of enoA), and        verified by PCR and DNA sequencing.        d) Establishment of a TopAct™ Compatible Hamster Model of CDAD

The hamster model of CDAD is a well-established model for the study oftoxin-induced antibiotic-associated diarrhea and colitis. The mostextensively studied model of C. difficile infection in hamsters is theprimary challenge model. Briefly, hamsters are pretreated withclindamycin (10-30 mg/kg) orogastrically 24 hours prior to theadministration of C. difficile spores to disrupt the normal colonicflora in the hamster. C. difficile spores (e.g. 100 spores of strain 630or B1; see Goulding et al., Infect Immun 2009; 77(12):5478-5485) areadministered orogastrically, and the hamsters are observed (CDAD primarychallenge model). Typically, 100% of hamsters succumb to disease between36 and 72 hours after spore administration. Prior to mortality, symptomsof disease include diarrhea and weight loss. In general, the symptomsare much more severe than those seen in humans, but the hamster modelresponds to therapeutic maneuvers used in clinical disease, such astreatment with vancomycin, and is therefore widely used in the study ofCDAD.

In order to simulate CDAD relapse control, a modified primary diseasehamster model is used. Vancomycin protects hamsters from C. difficiledisease, as it does in humans. When vancomycin treatment isdiscontinued, hamsters relapse with severe disease, but the attack ratevaries. Briefly, hamsters are given a single dose of clindamycinfollowed by the orogastric administration of C. difficile strain B1spores 1 day later. Vancomycin treatment began on the day of sporechallenge or 24 hours later and continued daily for two to foursubsequent days (CDAD relapse model). This protocol can be furtheroptimized to ensure relapse after rescue with vancomycin.

To assess the benefit of toxin-neutralizing Fab, intestinally deliveredby L. lactis, in preventing mortality in the primary and/or relapsemodel of infection in the hamster, it is important to document theimpact of clindamycin and vancomycin on the growth and Fab productioncapacity of L. lactis. Therefore, in vitro/in vivo studies are performedto determine viability and metabolic activity of the Fab-secreting L.lactis strains.

-   -   In vitro evaluation: Fab production (via ELISA) and growth (via        plating) from clindamycin/vancomycin-supplemented L. lactis        cultures is compared to an antibiotics-free culture and to a        culture supplemented with chloramphenicol (Cm) at 5 μg/ml. At        this concentration, Cm is a known inhibitor of protein synthesis        and growth, to which L. lactis is sensitive.    -   In vivo evaluation: Fab production (via ELISA) and viability        (via plating) is determined in the small/large intestine        following oral gavage of hamsters and concomitant treatment with        different doses of clindamycin/vancomycin.

These evaluations allow designing and adapting the hamster model(challenge and relapse) of CDAD that is well-suited for the evaluationof the L. lactis delivery system for their preventive and curativeeffects: this is using (lower) clindamycine (and vancomycine)concentrations and/or a different cocktail of antibiotics demonstratingsusceptibility to C. difficile infection without negative effect on L.lactis viability and metabolic activity.

e) Validation of the Hamster Model of CDAD.

In the hamster primary challenge model, Syrian golden hamsters (70 to 80g) are given different oral doses (qd, bid or tid) of the selectedanti-toxin A/B-secreting L. lactis (anti-toxin A and anti-toxin B aloneand combined) or 1 ml anti-toxin A/B mAb (as a positive control)intraperitoneally for 4 days beginning 3 days prior to theadministration of C. difficile spores. Clindamycin (dose or anothercocktail of antibiotics as defined above) is administered orogastrically24 hours prior to C. difficile spore challenge using a standard smallanimal feeding needle. Animals are observed for morbidity and mortality,intestinal tissues are scored for histological and macroscopic damage,and C difficile toxin production in luminal contents and feces isdetermined.

In the hamster relapse model, Syrian golden hamsters (70 to 80 g) aregiven clindamycin (dose or another cocktail of antibiotics as definedabove) orogastrically and 24 hours later challenged with C. difficile B1spores orogastrically. At the time of spore administration or 24 hourslater, vancomycin treatment (dose as defined above) orogastricallystarts and continues daily for a total of 2-4 days. Beginning 1, 2, 3, 4or 5 days following vancomycin treatment, different oral doses (qd, bidor tid) of the selected anti-toxin A/B-secreting L. lactis (anti-toxin Aand anti-toxin B alone and combined) or 1 ml anti-toxin A/B mAb (as apositive control, intraperitoneally) is administered for a total of 5-10days. Animals are observed for morbidity and mortality, intestinaltissues are scored for histological and macroscopic damage, and C.difficile toxin production in luminal contents and feces is determined.

It can be concluded that the gram-positive bacteria according to theinvention can effectively be used for immunizing against CDAD. Inparticular, the gram-positive bacteria according to the invention canprevent the occurrence of CDAD, prevent relapse of CDAD, as well astreat CDAD.

Example 14 Mouth Rinse Powder for Reconstitution

The Drug Substance (DS) of a mouth rinse formulation is a homogeneous,lyophilized powder of an engineered strain according to the inventionand mixed with cryoprotectants (dextrin, sorbitol and sodium glutamate).

The production process for the Drug Substance includes the followingsuccessive steps: fermentation, biomass concentration (bydiafiltrationor centrifugation), formulation with cryoprotectants,filling into suitable trays and bulk lyophilization. Homogenization andsieving of the lyophilized cake is performed to produce a homogeneouspowder (the Drug Substance) suitable for mixing with excipient andfilling into the desired pharmaceutical dosage form.

The mouth rinse Drug Product (DP) powder for reconstitution consists ofthe freeze-dried L. lactis bacteria, mixed with mannitol as an excipientand presented as a (compressed) powder. The clinical formulation is anoral, topical administration in the form of a mouth rinse. This mouthrinse suspension is prepared by reconstitution of the DP into a selectedsolution.

The production process of the mouth rinse powder for reconstitutionincludes a series of successive steps:

-   -   mixing of Bulk DS with mannitol,    -   compressing of 500 mg the DP powder mix in 500 mg dispersible        powder compacts,    -   filling of the compressed powder in glass vials,    -   closing of the vials with tamper-evident, child-resistant screw        caps, and    -   packaging of vials in aluminum (Alu) bags.

Example 15 Bicistronic Expression of CDP870

Dual cistron expression constructs were generated with heavy chain andlight chain of CDP870 anti-TNF Fab. All expression units are located onthe bacterial chromosome.

FIG. 15 A, schematic overview of CDP870 anti-TNF expression units invarious strains: CDP870 light and heavy chain Fab fusions to usp45secretion leader encoding sequences (SS::CDP870 VLCL and SS::CDP870VHCH1) were inserted as a second and third cistron downstream from usp45(sAGX0309, sAGX0319), enoA (sAGX0275) and gapB (sAGX0323, sAGX0326). Inthese strains, rpmD was used to couple SS::CD870 genes to usp45, enoA orgapB respectively. To avoid genetic instability, light and heavy chaingenes were coupled through the intergenic region preceding rplN. InFIGS. 15 B and C 4 identical clones of sAGX0326 (clone 1-4) wereanalyzed and reported. Strains were processed in parallel throughout theexperiments.

For the visualization and quantification of CDP870 anti-hTNF secretion,strains were inoculated from single colony into 10 ml GM17T (Difco™M17,BD, Sparks, MD, +0.5% glucose+200 μM thymidine) and grown for 16 hoursat 30° C. Bacteria from these saturated overnight cultures werecollected by centrifugation at 3220×g for 10 minutes and resuspended in10 ml fresh GM17T medium and grown for 2 hours at 30° C. Bacteria andcrude culture supernatants were separated by centrifugation at 3220×gfor 10 minutes. Crude supernatants from all strains were prepared inparallel and split up per strain for analysis (FIGS. 15 B and C).

Total protein content of 5 ml volumes of crude culture supernatants wasextracted with phenol, precipitated with ethanol and resuspended inSDS-PAGE sample buffer.

Equivalents of 1 ml of crude culture supernatants were analyzed bywestern blot using goat anti-human Fab as a primary antiserum andrevealed by rabbit anti-goat AP and NBT/BCIP staining (FIG. 15 B;strains are indicated at the right of respective lanes).

Crude supernatants from strains carrying the individual constructs wereassayed for the presence of hTNF binding activity. This was done bydirect ELISA using hTNF as capture protein with Cimzia as a referencestandard. VLCL portions were detected by goat anti-human IgG antiserumand revealed by horseradish peroxydase (HRP) conjugated anti-goatantiserum. HRP activity was measured by colorimetric assay. Data arepresented in FIG. 15 C, strains are indicated underneath respectivebars.

FIGS. 15 (B and C) reveal that both heavy chain and light chains werehighly expressed by the dual cistron constructs, leading to high levelsof functional CDP870 anti-TNF Fab. FIGS. 15 (B and C) reveals thatCDP870 anti-TNF expression slightly increased when heavy and light chaingenes were inserted as a second and third cistron downstream from enoAwhen compared to insertion downstream of usp45. FIGS. 15 (B and C)further reveals that CDP870 anti-TNF expression was substantiallyincreased when heavy and light chain genes were inserted as a second andthird cistron downstream from gapB when compared to insertion downstreamof usp45 or enoA.

For the determination of specific hTNF neutralizing capacity (biologicalactivity per quantity of TNF binding protein), strains were inoculatedfrom single colony into 5 ml GM17T and grown for 16 hours at 30° C.Bacteria from these saturated overnight cultures were collected bycentrifugation at 3220×g for 10 minutes and resuspended in 5 ml BM9Tmedium and grown for 2 hours at 30° C. Bacteria and crude culturesupernatants were separated by centrifugation at 3220×g for 10 minutes.Crude supernatants from strains carrying the individual strains wereprepared in parallel and split up per strain for analysis (FIGS. 15 Dand E).

Crude supernatants from strains carrying the individual constructs wereassayed for the presence of TNF binding activity. This was done bydirect ELISA using human TNF as capture protein with Cimzia as areference standard. VLCL portions were detected by goat anti-human IgGantiserum and revealed by horseradish peroxydase (HRP) conjugatedanti-goat antiserum. HRP activity was measured by colorimetric assay.All strains were treated in parallel. Data are presented in FIG. 15 D,strains are indicated underneath respective bars.

Crude supernatants from strains carrying the individual constructs wereassayed for the presence of TNF neutralizing activity. This was done byincubation of hTNF susceptible WEHI cells with human TNF and addition ofanti-TNF. Anti-TNF will scavenge hTNF and will protect WEHI cells fromcell death. A ½ dilution series of the crude supernatants as well asreference standard (Cimzia at 63 ng/ml) were added to the cell culturessubjected to hTNF. The impact on cell death was determined. Data arepresented in FIG. 15 E, strains are indicated underneath respectivebars.

FIG. 15 D reveals that both heavy chain and light chains were highlyexpressed by the dual cistron constructs, leading to high levels offunctional CDP870 anti-TNF Fab. FIG. 15 D further reveals that CDP870anti-TNF expression substantially increased when heavy and light chaingenes were inserted as a second and third cistron downstream from gapBwhen compared to insertion downstream of usp45.

FIGS. 15 D and E show that specific TNF neutralizing capacity(biological activity per quantity of TNF binding protein) of CDP870anti-TNF in the crude culture supernatants of strains sAGX0323 andsAGX0326 is identical to that of Cimzia.

Example 16 Efficacy of L. lactis Secreting Anti-hTNFα Fab Fragment

The experimental set up is based on the Tg1278TNFko mouse, a transgenicmouse with normally regulated human TNF expression in the absence ofmouse TNF (Keffer et al. EMBO. J 10, 4025-4031, 1991). Colitis wasinduced by rectal administration challenge of 4% TNBS in 40% ethanolafter one cutaneous presensitization. Briefly, mice were sensitized 7days (Day −7) prior intrarectal challenge by applying 1 volume 5% TNBS+4volumes 4:1 acetone:olive oil to a shaved 1.5×1.5 cm skin area on theback. On the day of the challenge (Day 0), mice were first anesthetizedwith ketamine/xylazine, subsequently 100 μl 4% TNBS/40% EtOH wasadministered per rectum by a flexible catheter inserted 4 cm into therectum. To ensure equal distribution of the enema within the colon, micewere held in a vertical position for 30 seconds directly after therectal challenge.

Treatment was initiated 1 day before the rectal TNBS challenge (Day −1)and was continued for another 4 days (Day +3). Three groups of micereceived once daily intragastric inoculations with 10¹⁰ CFU of MG1363(negative control), 10¹⁰ CFU sAGX0309, or 10 μg Cimzia (positivecontrol). Starting from Day 0 and on a daily basis, mice were monitoredfor body weight, morbidity and survival. On Day +3 mice were sacrificedand colon samples and serum were collected for histology (colon) andcytokine (colon and serum) analysis.

Treatment with a strain according to an embodiment of the invention(anti-hTNF-secreting L. lactis strain sAGX0309) resulted in an enhancedsurvival (FIG. 16 and Table 7), in comparison with the wild type L.lactis strain MG1363 and surprisingly even a higher survival percentagethan mice treated with Cimzia.

TABLE 7 L. lactis L. Lactis SURVIVAL sAGX0309 MG1363 Cimzia Day 1 100% 100%  100%  (7/7) (9/9) (9/9) Day 2 86% 89% 89% (6/7) (8/9) (8/9) Day 386% 56% 78% (6/7) (5/9) (7/9)

Body weight of the mice was also followed during treatment, and isdepicted in FIG. 17. From FIG. 17, it is evident that weight loss islower after treatment with a strain according to an embodiment of theinvention in comparison with treatment with Cimzia.

The histological status of the colon was also analyzed and ahistological score was attributed according to Table 8. The results areindicated in FIG. 18. From FIG. 18, a significant improvement in thehistological score, and hence a diminished colitic pathology, is evidentafter treatment with a strain according to an embodiment of theinvention.

TABLE 8 Histological score Description 0 No inflammation; no epithelialdammage 1 Inflammation in the mucosa around the crypt bases; noepithelial dammage 2 Inflammation in the submucosa; mild epithelialdammage with loss of goblet cells 3 Inflammation in the submucosa; localloss of crypt architecture 4 Inflammation in the submucosa; loss ofcrypt architecture in extended areas of the mucosa

Finally, from FIG. 19, it is apparent that treatment with a strainaccording to an embodiment of the invention resulted in a suppression ofcolonic proinflammatory cytokine secretion.

The invention claimed is:
 1. A pharmaceutical composition comprising agram-positive bacterium comprising a polycistronic expression unit,wherein said polycistronic expression unit comprises a functionalendogenous gene and one or more exogenous genes encoding a therapeuticpolypeptide, wherein said endogenous gene and said one or more exogenousgenes are transcriptionally controlled by a promoter endogenous to saidgram-positive bacterium, wherein said promoter (i) is a ribosomal genepromoter or a glycolysis gene promoter, (ii) is the native promoter ofsaid endogenous gene, and (iii) said promoter and said endogenous geneare located in their native chromosomal locus in the gram-positivebacterium, and wherein said endogenous gene is transcriptionally coupledto said one or more exogenous genes by chromosomal integration of saidone or more exogenous genes into said locus.
 2. The pharmaceuticalcomposition of claim 1, wherein said promoter is selected from the groupconsisting of the promoters of eno, usp45, gap, pyk, rpmB and rplS ofsaid gram-positive bacterium.
 3. The pharmaceutical composition of claim2, wherein said one or more exogenous genes is transcriptionally coupledto the 3′ end of said endogenous gene.
 4. The pharmaceutical compositionof claim 3, wherein said one or more exogenous genes is the most 3′ geneof said polycistronic expression unit.
 5. The pharmaceutical compositionof claim 1, wherein said endogenous gene and said one or more exogenousgenes are transcriptionally coupled by intergenic region or regionsactive in said gram-positive bacterium.
 6. The pharmaceuticalcomposition of claim 5, wherein said intergenic region or regions isendogenous to said gram-positive bacterium.
 7. The pharmaceuticalcomposition of claim 5, wherein said intergenic region is selected fromthe group consisting of intergenic regions preceding rplW, rplP, rpmD,rplB, rpsG, rpsE, rplN, rplM, rplE, and rplF.
 8. The pharmaceutical ofclaim 1, wherein said therapeutic polypeptide is present in atherapeutically effective amount.
 9. The pharmaceutical composition ofclaim 1, wherein said therapeutic polypeptide has a preventive effect ina subject.
 10. The pharmaceutical composition of claim 1, wherein saidpolypeptide is an antigen for inducing immunity or immunotolerance, anon-vaccinogenic therapeutically active polypeptide, or an antibody or afunctional fragment thereof.
 11. The pharmaceutical composition of claim10, comprising a functional fragment of an antibody, wherein saidfunctional fragment is an Fab fragment.
 12. The pharmaceuticalcomposition of claim 1, wherein said one or more exogenous genes are 3′of said endogenous gene in said locus.
 13. The pharmaceuticalcomposition of claim 1, wherein one of said one or more exogenous genesencodes a light chain (V_(L)) of an antibody or a functional fragmentthereof, and another of said one or more exogenous genes encodes a heavychain (V_(H)) of said antibody or a functional fragment thereof.
 14. Thepharmaceutical composition of claim 13, wherein said functional fragmentis an Fab fragment.
 15. The pharmaceutical composition of claim 13,wherein said exogenous gene encoding V_(L) or functional fragmentthereof is transcriptionally coupled to the 3′ end of said exogenousgene encoding V_(H) or functional fragment thereof.
 16. Thepharmaceutical composition of claim 1, wherein said gram-positivebacterium is a lactic acid bacterium or a Bifidobacterium.
 17. Thepharmaceutical composition of claim 16, wherein said lactic acidbacterium is a Lactococcus, a Lactobacillus, or an Enterococcusbacterium.
 18. The pharmaceutical composition of claim 17, wherein saidlactic acid bacterium is Lactococcus lactis or Enterococcus faecium. 19.The pharmaceutical composition of claim 1, further comprising apharmaceutically acceptable carrier.
 20. A method of delivering atherapeutic polypeptide to a human or animal subject comprisingadministering to said human or animal subject the pharmaceuticalcomposition of claim 1, whereby said administration is effective todeliver said therapeutic polypeptide.
 21. The pharmaceutical compositionof claim 1, wherein said endogenous gene is a full length endogenousgene.
 22. The pharmaceutical composition of claim 1, wherein saidpromoter is gapB, and wherein said gapB promoter is coupled to a geneencoding Human Trefoil Factor 1 (hTFF1) by an intergenic regioncomprising rpmD.
 23. The pharmaceutical composition of claim 1, whereinsaid promoter is usp45, and wherein said usp45 promoter is coupled to agene encoding Human pro-insulin (ins) by an intergenic region comprisingrpmD.
 24. The pharmaceutical composition of claim 1, wherein saidpromoter is enoA, and wherein said enoA promoter is coupled to a geneencoding Human pro-insulin (ins) by an intergenic region comprisingrpmD.
 25. The pharmaceutical composition of claim 10, wherein saidpolypeptide is a human pro-insulin, a human trefoil factor 1 (hTFF1), ahuman interleukin-10 (hIL-10), a human interleukin-27 (hIL-27), a cA2anti-hTNF Fab or a CDP870 anti-TNF Fab.
 26. The pharmaceuticalcomposition of claim 2, wherein said one or more exogenous genes aretranscriptionally coupled by intergenic region or regions active in saidgram-positive bacterium.
 27. The pharmaceutical composition of claim 26,wherein said intergenic region is selected from the group consisting ofintergenic regions preceding rplW, rplP, rpmD, rplB, rpsG, rpsE, rplN,rplM, rplE, and rplF.
 28. The pharmaceutical composition of claim 1,wherein the therapeutic polypeptide is selected from the groupconsisting of: a cytokine, a growth factor, and a wound healing factor.29. The pharmaceutical composition of claim 10, wherein the antibody isselected from the group consisting of: a chimeric antibody, a dAb, abispecific antibody, a trispecific antibody, a multispecific antibody, abivalent antibody, a multivalent antibody, a nanobody, an Fab', anF(ab')₂, an scFv, an Fv, an Fd, a diabody, a triabody, a single chainantibody, and a single variable domain.
 30. The pharmaceuticalcomposition of claim 1, wherein the therapeutic polypeptide is selectedfrom the group consisting of: IL-Ira, IL-10, IL-27, a trefoil peptide,an auto-antigen, an allergen, a gluten allergen, a brain derivedneurotropic factor, a ciliary neurotropic factor, IL-1, a colonystimulating factor, interferon-ω, transforming growth factor f3,insulin, a tissue plasminogen activator, a cytokine antagonist, aclotting factor, a hepatocyte growth factor, interferon α, alphaantitrypsin.